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Targeted therapy in gastrointestinal malignancies

Increased understanding of cancer pathogenesis has identified several pathways that serve as potential targets for novel targeted agents in development. The selection of targeted cancer therapy based on biomarkers has instigated a new era of personalized medicine and changed the way we practice oncology. Many targeted agents are approved for treatment of gastrointestinal malignancies most targeting tumor angiogenesis, and many more are in different phases of development. Here we briefly summarize nine different targeted agents that are approved currently in the U.S. and several other agents currently being studied in various gastrointestinal cancers.

INTRODUCTION
With improved understanding of cancer genomics, proteomics and molecular events associated with cell growth, proliferation, angiogenesis, apoptosis and signal transduction, along with biotechnological advancements such as immunohistochemical and hybridization techniques, our knowledge of cancer pathogenesis has increased exponentially. Though incomplete, we have identified several key molecular events involved in carcinogenesis and targeting them offers survival benefit in several cancers such as breast cancer, colon cancer and leukemia. The concept of targeted therapy was born more than a century ago but its practical application in cancer therapy took several decades. Today we have developed several agents designed to target specific molecule such as cell receptors, enzymes various cytokines and signaling pathways. The National Cancer Institute defines targeted therapy as "a type of treatment that uses drugs or other substances, such as monoclonal antibodies, to identify and attack specific cancer cells". [1] Over the last 2 decades, the development of drugs targeted at specific molecular pathway/receptor has led us one step closer to individualized cancer therapy. Many targeted therapies have been approved by the United States Food and Drug Administration (FDA) for clinical use in humans while as many are in various phases of development as well. For the scope of this review article, we will discuss only the relevant trials in gastrointestinal cancers with regard to each targeted agent. Figure 1 shows the conceptual diagram of several molecular targets and signaling pathways involved in carcinogenesis and Table 1 describes targeted agents studied in gastrointestinal (GI) cancers and their FDA approval status. and irinotecan (FOLFIRI) as the first line treatment for KRAS mutation negative and anti-epidermal growth factor receptor (EGFR) expressing metastatic colorectal cancer based on a randomized, multicenter, open-label, phase III trial (CRYSTAL trial) where each group had 599 patients (cetuximab + FOLFIRI vs. FOLFIRI alone). [2] The addition of cetuximab to FOLFIRI reduced the risk of progression by 15%, increased the median progression free survival (PFS) from 8 to 8.9 months (mo) (hazard ratio [HR]: 0.85, 95% confidence interval [CI]: 0.72-0.99, P = 0.048), increased the median overall survival (OS) from 18.6 to 19.9 mo (HR: 0.93, 95% CI: 0.81-1.07, P = 0.31). The adjusted odds ratio for a tumor response to cetuximab group was 1.40 (95% CI: 1.12-1.77, P = 0.004). Median OS in cetuximab + FOLFIRI and FOLFIRI groups were respectively 24.9 and 21.0 mo in the wild type-KRAS population as well as 17.5 and 17.7 mo, in the mutant-KRAS population.
Cetuximab has been studied in the gastroesophageal cancer with limited success. Cetuximab in combination with chemoradiation containing different regimen has shown up to 40% objective response rate in locally advanced disease and up to 16.6 mo of OS in metastatic disease. [3,4] The Southwest Oncology Group directed (S0205) phase III trial evaluated the role of cetuximab in metastatic or locally advanced unresectable pancreatic adenocarcinoma, where 371 patients were treated with gemcitabine alone versus 372 patients with gemcitabine + cetuximab. [5] The addition of cetuximab to gemcitabine failed to improve median OS and PFS significantly (one-sided P = 0.19 for OS and P = 0.18 for PFS). Moreover, EGFR expression had no impact on median survival (HR: 0.98, 95% CI: 0.83-1. 17, P = 0.42). The efficacy of gemcitabine + oxaliplatin + cetuximab was assessed in 45 unresectable or advanced hepatocellular carcinoma (HCC) patients in a multicenter phase II trial. Median PFS of 4.7 mo (95% CI: 2.6-9.5) and median OS of 9.5 mo (95% CI: 7.8-11) were observed. The 1-year survival rate was 40%. [6] An interim analysis of the BINGO trial, an open label phase II study comparing gemcitabine + oxaliplatin with or without cetuximab was presented at the 2009 American Society of Clinical Oncology meeting. At 4 mo, PFS rate was 61% (95% CI:  in cetuximab group compared to 44% (95% CI:  in the other group. [7] supportive care (HR: 0.76, 95% CI: 0.60-0.98); (2) median OS of 8.1 mo in wild-KRAS tumors randomized to panitumumab versus 4.4 mo in patients with mutant-KRAS tumors randomized to supportive care (HR: 0.56, 95% CI: 0.49-0.87); (3) median OS of 7.9 mo in wild-KRAS group versus 4.7 mo in all patients with mutant-KRAS tumors, regardless of treatment group assignment. [10] Panitumumab as a first line agent in metastatic colorectal cancer was evaluated in a phase III randomized  trial (PRIME) in combination with 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX4). In wild-KRAS tumors, the addition of panitumumab to FOLFOX4 significantly improved PFS compared with FOLFOX4 alone (9.6 mo vs. 8.0 mo, respectively; HR: 0.80, 95% CI: 0.66-0.97, P = 0.02). However, overall response rate was not significantly different (55% vs. 48% respectively, P = 0.068). [11] Adding panitumumab to irinotecan alone does not improve OS in wild-KRAS tumors. [12] Combination of panitumumab and FOLFIRI as a second line agent showed significant improvement in PFS compared to FOLFIRI alone (HR: 0.73, 95% CI: 0.59-0.9, P = 0.004). [13] Combination targeted therapies (panitumumab + bevacizumab) when combined with standard chemotherapy (oxaliplatin and irinotecan based) as a first line agent in metastatic colorectal cancer decreases PFS and increases toxicity. [14] Panitumumab, when studied with chemotherapy (gemcitabine + oxaliplatin + capecitabine) as a first line therapy for wild-KRAS expressing unresectable biliary cancer, median PFS of 8.3 mo (95% CI: 6.7-8.7) and median OS of 10 mo (95% CI: 7.4-12.7) were observed.
A randomized, double-blind, placebo controlled, phase III trial evaluated the addition of aflibercept to gemcitabine in patients with advanced pancreatic cancer. The study was stopped for futility following a planned interim analysis. [27] Nimotuzumab (Theracim ® , h-R3 ® ) Nimotuzumab, a humanized monoclonal antibody, binds to extracellular domain of EGFR, and inhibits downstream signal conduction. It is approved in more than 20 countries but not U.S. for various indications (i.e., gliomas, head and neck cancer, esophageal cancer, etc.) with very favorable side-effect profile. A retrospective study evaluated 66 esophageal squamous cell cancer patients treated with Nimotuzumab and radiation/chemoradiation. Median OS and PFS were 26.0 mo and 16.7 mo, respectively. OS, PFS and locoregional control at 2 years were 54%, 37% and 80%, respectively. [28] A phase II trial evaluated nimotuzumab 200 mg weekly as a second line agent in advanced pancreatic cancer patients. For 36 evaluable patients, median PFS of patients with stable disease was 19.2 weeks and 6.7 weeks for all patients (95% CI: 6.43-7.14). PFS after 1 year was 10.3% with median OS of 18.1 weeks. [29] Yttrium clivatuzumab tetraxetan (hPAM4-Cide ® ) A monoclonal antibody, clivatuzumab, combined with yttrium (a radioisotope) where tetraxetan acts as a chelator (YCT). YCT is directed to mucin antigen found in 85% pancreatic adenocarcinoma but absent in normal pancreatic tissue. [30] This unique combo was studied along with gemcitabine, a radio sensitizer, in advanced pancreatic cancer based on preclinical data. Previously untreated 38 patients (n = 33, stage IV; n = 6, stage III) where 19 patients received escalating dose of YCT in repeat cycles. Grade III/IV toxicities were observed in all patients after second cycle. The median OS was 7.7 mo for all 38 patients, including 11.8 mo for those who received repeated cycles (46% [6 of 13 patients] ≥1 year), with improved efficacy at higher doses. [31] Conatumumab (AMG 655) Conatumumab, an agonist monoclonal antibody directed towards the death receptor 5, causing the activation of capcase and subsequent apoptosis. Patients with mutant KRAS metastatic colorectal cancer refractory to fluoropyrimidine-and oxaliplatin-based chemotherapy were randomized to FOLFIRI plus conatumumab, ganitumab, or placebo. Median PFS in conatumumab, ganitumab, and placebo group were 6.5 mo (HR: 0.69; P = 0.147), 4.5 mo (HR: 1.01; P = 0.998) and 4.6 mo, respectively. median OS were 12.3 mo (HR: 0.89; P = 0.650), 12.4 mo (HR: 1.27; P = 0.357) and 12.0 mo, respectively. [32] In metastatic pancreatic adenocarcinoma, addition of conatumumab to gemcitabine showed trend towards improved 6 mo survival rate compared to gemcitabine alone (59% vs. 50%). [33] Ganitumab (AMG 479) Ganitumab, a monoclonal antibody directed against insulin-like growth factor-1. In a multicenter, phase II, open-label trial, ganitumab was studied in metastatic progressive carcinoid or pancreatic neuroendocrine tumor (PNET) but failed to show any significant tumor response. [34] However, in metastatic pancreatic adenocarcinoma, addition of ganitumab to gemcitabine showed trend toward improved 6 mo survival rate compared to gemcitabine alone (57% vs. 50%). [33] As mentioned earlier, Journal of Carcinogenesis A peer reviewed journal in the field of Carcinogenesis and Carcinoprevention ganitumab + FOLFIRI in metastatic colorectal cancer failed to show any significant tumor response.

Regorafenib (Stivarga ® )
Regorafenib is a small molecule multikinase inhibitor. It is approved for patients with metastatic colorectal cancer who have been previously treated with fluoropyrimidine-, oxaliplatin-and irinotecan-based chemotherapy, an anti-VEGF therapy and if KRAS wild type, an anti-EGFR therapy based on the results of a placebo controlled, randomized, phase III trial (CORRECT trial). [60] In this study, median OS was 6·4 mo in regorafenib group (n = 500) versus 5·0 mo in placebo group (n = 253) (HR: 0·77, 95% CI: 0·64-0·94, one-sided P = 0·0052). Regorafenib is also approved for patients with locally advanced, unresectable or metastatic GIST who have been previously treated with imatinib and sunitinib. Efficacy was confirmed in a phase III trial in which 199 patients were randomly assigned to best supportive care with either regorafenib (160 mg daily for 3 of every 4 weeks) or placebo. Median PFS was 4·8 mo (range 1·4-9·2) for regorafenib and 0·9 mo (range 0·9-1·8) for placebo (HR: 0·27, 95% CI: 0·19-0·39, P < 0·0001). [61] In a phase II trial of regorafenib in HCC after failure of first line sorafenib, 36 patients were included. Median TTP and OS were 4.3 mo and 13.8 mo respectively, but 89% patients had treatment interruption due to adverse drug reaction. [62] Gefitinib (Iressa ® ) Gefitinib, an EGFR tyrosine kinase inhibitor, when studied in phase II trials in metastatic or recurrent esophageal/ gastroesophageal junction adenocarcinoma and squamous cell cancers, has been well tolerated. Gefitinib increases the radiosensitivity of gastric cancer cell lines. [63] In pancreatic cell lines, gefitinib has shown to reverse multidrug resistance via RAF1/ERK signaling pathway. [64] Moreover, gefitinib has shown antiproliferative effect on pancreatic cancer cell lines as well as on cholangiocarcinoma cells and gallbladder carcinoma when combined with gemcitabine. In a phase II trial of gefitinib combined with gemcitabine, 6 mo PFS was 30%. Median PFS and OS were 4.1 and 7.3 mo, respectively. [65] In human and murine HCC cells, gefitinib induced cell cycle arrest, apoptosis and cell growth inhibition. [66,67] In phase II trials, FOLFOX4 + gefitinib showed median OS of 12 mo and median event free survival of 5.4 mo in previously treated metastatic colorectal cancer, [68] as well as median OS of 20.5 mo and median TTP of 9.3 mo in previously untreated metastatic cases. [69] These results discourage the addition of gefitinib to FOLFOX-4 in metastatic colorectal cancer. In chemotherapy naïve metastatic colorectal cancer, addition of gefitinib to capecitabine + oxaliplatin showed 80% disease control rate, median TTP was 7.3 mo (95% CI: 4.76-9.2) and median OS was 21.9 mo (95% CI: 15.1-not reached). [70] EGFR phosphorylation status in colon cancer is predictive of response to gefitinib, even synergistic when used with platinum based chemotherapy. [71] Expression of p21 gene in combination with p53 gene mutation is a predictor of resistance to combination of chemotherapy + gefitinib. [72] The combination of gefitinib + cetuximab has synergistic effect on cell proliferation and apoptosis when studied in colon cancer cell lines. [73] Cediranib (AZD 2171) Cediranib, a vascular EGFR tyrosine kinase inhibitor, inhibits tumor cell migration and invasion, with no effects on cell proliferation when studied in colorectal, pancreatic and HCC cell lines. [74] Cediranib, when studied in advanced HCC, median OS of 5.8 mo (95% CI: 3.4-7.3) and median TTP of 2.8 mo (95% CI: 2.3-4.4) were observed. Grade ≥ 3 adverse events were observed in 93%. [75] In a phase III, randomized, double blind trial (HORIZON II), previously untreated metastatic colorectal cancer patients received either cediranib (n = 502) or placebo (n = 358) in addition to FOLFOX or capecitabine + oxaliplatin. The addition of cediranib to FOLFOX or capecitabine + oxaliplatin resulted in median PFS prolongation from 8.3 mo in placebo arm to 8.6 mo in cediranib arm (HR: 0.84, 95% CI: 0.73-0.98, P = 0.0121) but had no impact on OS (median OS, 19.7 mo for cediranib vs. 18.9 mo for placebo, HR: 0.94; 95% CI: 0.79-1.12, P = 0.5707). There were no significant differences in objective response rate, duration of response, or liver Journal of Carcinogenesis A peer reviewed journal in the field of Carcinogenesis and Carcinoprevention resection rate. [76] In a phase II, randomized trial of cediranib in metastatic colorectal cancer, 20 mg daily dose reached primary objective, median PFS increased from 8.3 mo to 10.2 mo (HR: 0.70, 95% CI: 0.44-1.11, P = 0.167). [77] Cediranib was comparable to bevacizumab when combined with modified FOLFOX6 regimen (HORIZON III trial). [78] Lapatinib (Tykerb ® ) Lapatinib, a dual tyrosine kinase inhibitor associated with HER2/neu and EGFR receptor. The combination of lapatinib with cisplatin or 5-FU or trastuzumab synergistically inhibits cell proliferation and exhibits an enhanced pro-apoptotic effect on esophageal cancer cells. Lapatinib has not proved to be beneficial in HCC, pancreatic, biliary or refractory colorectal cancers when studied in small preclinical or phase II studies.

Vandetanib (Caprelsa ® )
Vandetanib inhibits tyrosine kinase activity of EGFR, vascular EGFR families, RET, BRK and TIE2. In a preclinical trial, vandetanib synergistically enhanced the sunitinib-associated inhibition of gastric cancer cell growth. [79] Preclinical trial has shown beneficial effect of vandetanib in liver and early intestinal cancer in mice. In several phase I trials, vandetanib is being studied with different chemotherapy combination in advanced colorectal cancer.

Linifanib (ABT 869)
Linifanib is a selective inhibitor of vascular EGFR and platelet-derived growth factor receptor (GFR) tyrosine kinases. In a phase II trial of unresectable or metastatic HCC, linifanib showed median time to disease progression of 3.7 mo and median OS of 9.7 mo and thus raised hopes for another effective targeted agent in HCC beyond sorafenib. [80] Vatalanib (PTK787) Vatalanib is an oral antiangiogenic agent that acts as a vascular EGFR inhibitor. In a phase III, randomized, placebo controlled trial of previously untreated metastatic colorectal cancer, patients were randomly assigned to FOLFOX4 + vatalanib (evaluable n = 581) or FOLFOX4 + placebo (evaluable n = 575) arm. [81] Median PFS in vatalanib arm was 7.7 mo versus 7.6 mo in placebo arm (HR: 0.88, 95% CI: 0.74-1.03, P = 0.118); median OS in vatalanib and placebo arms were respectively 21.4 mo and 20.5 mo (HR: 1.08, 95% CI: 0.94-1.24, P = 0.260). In post hoc analysis of PFS in patients (n = 158/arm) with high serum lactate dehydrogenase, a potential marker of hypoxia, PFS was longer with vatalanib versus placebo (7.7 vs. 5.8 mo, respectively; HR: 0.67, 95% CI: 0.49-0.91, P = 0.009). Similar results were observed in another phase III, randomized trial when FOLFOX4 + vatalanib or placebo combination was studied in previously treated metastatic colorectal cancer. [82] Subsequently, vascular density analysis was performed in biopsy specimens of 141 colorectal cancer patients (placebo arm [n = 70] and vatalanib [n = 71]) in above phase III trials. The vascular density correlated with response to therapy, PFS and OS. The response rate increased from 15% (3/20) to 50% (11/22) in tumors with high vascular density, when vatalanib was added to chemotherapy (P = 0.02). [83] Brivanib (BMS 582664) Brivanib is an inhibitor of vascular EGFR and fibroblast GFR. The results of large phase III trials are disappointing despite encouraging phase II trials, where brivanib was used as a first and second line agent in advanced HCC. In a phase III, multicenter, double blind, placebo controlled, randomized trial of advanced HCC (BRISK-PS), patients failed/intolerant to sorafenib, were randomly assigned to receive brivanib and best supportive care (n = 263) versus placebo and best supportive care (n = 132). [84] Median OS was 9.4 mo for brivanib and 8.2 mo for placebo (HR: 0.89, 95.8% CI: 0.69-1.15, P = 0.3307) and median TTP was 4.2 mo for brivanib and 2.7 mo for placebo (HR: 0.56, 95% CI: 0.42-0.76, P < 0.001). Moreover, brivanib related side-effect caused discontinuation in 23%. In another, phase III, randomized, double blind trial, brivanib (n = 577) and sorafenib (n = 578) were compared head to head in untreated advanced HCC (BRISK-FL). The primary end point of OS non-inferiority for brivanib versus sorafenib in the per-protocol population (n = 1150) did not meet (HR: 1.06, 95.8% CI: 0.93-1.22). Median OS was 9.9 mo for sorafenib and 9.5 mo for brivanib. TTP, objective response rate and disease control rate were similar between two arms. [85] The combination of cetuximab and brivanib has been studied in metastatic, chemotherapy refractory, wild type-KRAS colorectal cancer in a phase III, randomized, placebo controlled trial (AGITG CO.20). Median OS in the intent-to-treat population was 8.8 mo in brivanib arm (n = 376) and 8.1 mo in placebo arm (n = 374) (HR: 0.88; 95% CI: 0.74-1.03, P = 0.12). Median PFS was 5.0 mo in brivanib and 3.4 mo in placebo arm (HR: 0.72, 95% CI: 0.62-0.84; P < 0.001). Any grade ≥ 3 adverse events were 78% in brivanib and 53% in placebo arms. [86] Nilotinib (Tasigna ® ) Nilotinib, a second generation tyrosine kinase inhibitor, prevents autophosphorylation of c-KIT and platelet GFR. In a phase III, open label trial, nilotinib was studied in advanced GIST resistant to imatinib and sunitinib. [87] Patients (n = 248) were randomized to nilotinib or best supportive care with imatinib/sunitinib/placbo. Median PFS was similar in both arms (nilotinib 109 days vs. best supportive care 111 days; P = 0.56) based on central radiology review. However, intent-to-treat analysis favored nilotinib over best supportive care (119 days vs. 70 days, P = 0.0007). A nonsignificant but positive trend was noted for OS in nilotinib group. Post

Journal of Carcinogenesis 9
A peer reviewed journal in the field of Carcinogenesis and Carcinoprevention hoc subset analyses in patients with progression and only one prior regimen each of imatinib and sunitinib revealed a significant difference in median OS of > 4 months in favor of nilotinib (405 vs. 280 days; P = 0.02).

Everolimus (Afinitor ® )
Everolimus is a mammalian target of rapamycin inhibitor. Everolimus causes dose dependent decrease in cell proliferation, cell cycle arrest in G1/S phase and damages cell shape. [88] Everolimus with capecitabine showed favorable toxicity profile and modest benefit (median PFS 1.8 months [95% CI: 0.8-2.8] in previously treated metastatic gastric cancer). [89] Everolimus monotherapy in previously chemotherapy treated advanced gastric cancer was studied in a phase II trial where no complete or partial response were observed. However, a decrease in tumor size from baseline was observed in 45%, disease control rate was 56.0% (95% CI: 41.3-70.0), median PFS was 2.7 mo (95% CI: 1.6-3.0) and median OS was 10.1 mo (95% CI: 6.5-12.1). [90] Everolimus augments the effects of sorafenib synergistically in orthotopic HCC model. [91] In a phase II trial of everolimus in advanced HCC with 0-2 previous regimens, median PFS and OS were 3.8 mo (95% CI: 2.1-4.6) and 8.4 mo (95% CI: 3.9-21.1), respectively. The estimated PFS rate at 24 weeks was 28.6% (95% CI: 7.9-49.3). [92] In a phase III, randomized trial, low-grade or intermediate-grade PNETs with radiologic progression within the previous 12 mo to receive everolimus (n = 207), or placebo (n = 203). Median PFS was 11.0 mo in everolimus versus 4.6 mo in placebo (HR: 0.35, 95% CI: 0.27-0.45, P < 0.001), representing a 65% reduction in the estimated risk of progression or death. [93] Everolimus and octreotide are effective independently in PNETs. However, this combination was recently studied in a phase II trial in treatment naïve 50 patients with well differentiated metastatic neuroendocrine tumors. Disease control rate was 92% and median TTP was 16.3 mo (95% CI: 10.7-20.1). [94] Everolimus is ineffective in metastatic colorectal cancer resistant to multiple chemotherapy agents. [95] Selumetinib (AZD 6244) Selumetinib inhibits mitogen-activated protein kinase (MEK or MAPK/ERK kinase) 1 and 2. Single agent (selumetinib or capecitabine) trial in colorectal cancer patients who previously failed one or two chemotherapeutic agent showed similar median PFS (81 days in selumetinib vs. 88 days in capecitabine arm). [96] In a phase II trial, erlotinib + selumetinib combination was studied in pancreatic adenocarcinoma (n = 46) treated with one prior chemotherapy. Estimated median PFS and OS by Kaplan-Meier were 2.6 and 7.5 mo, respectively and disease control rate was 51% in 41 evaluable patients. [97] Combination of selumetinib and vorinostat synergistically inhibits cell proliferation in two colorectal cancer cell lines with KRAS mutation. [98] Another phase II trial compared selumetinib or capecitabine as a single agent in advanced or metastatic pancreatic cancer patients (n = 70) who failed gemcitabine. Median OS was 5.4 mo in selumetinib arm and 5.0 mo in capecitabine arm (HR: 1.03, two-sided 80% CI: 0.68-1.57, P = 0.92). [99] In a phase II study of selumetinib in metastatic biliary cancer, median PFS and OS were 3.7 (95% CI: 3.5-4.9) and 9.8 (95% CI: 5.97-not available) mo, respectively. [100] Trametinib (Mekinist ® ) Trametinib is a reversible MEK 1 and 2 inhibitor. In a pre-clinical study of pancreatic cancer cell lines treated with trametinib, cancer cell proliferation was inhibited and addition of EGFR/HER2 inhibitor, lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. [101] Trametinib when combined with 5-FU markedly decreased colony numbers of colon cancer cell lines. [102]

CLINICALLY USED BIOMARKERS FOR TARGETED THERAPY SELECTION IN GI CANCERS
In GI cancers, tumor receptor tyrosine kinase-targeted therapies (i.e., trastuzumab, imatinib) and antibodies (cetuximab, panitumumab) demonstrate a robust clinical response in patients that exhibit overexpression of their intended targets or certain genetic alterations. Trastuzumab is used only in gastric or gastroesophageal junction cancers that overexpress HER2. Imatinib is used for GIST expressing c-KIT and further studies of KIT mutation and platelet derived growth factor mutations have also been shown to correlate with response/resistance to imatinib. Although initially approved for all metastatic colon cancers, the label was changed to indicate cetuximab should only be used for the treatment of KRAS mutation-negative (wild-type), EGFR-expressing metastatic colorectal cancer (in combination with FOLFIRI as first-line treatment, in combination with irinotecan [in patients refractory to irinotecan-based chemotherapy], or as a single agent in patients who have failed oxaliplatin and irinotecan based chemotherapy or who are intolerant to irinotecan). Similarly, monotherapy with panitumumab in treatment of EGFR-expressing refractory metastatic colorectal cancer with disease progression on or following fluoropyrimidine-, oxaliplatin-and irinotecan-based regimens. Currently, no biomarkers for antiangiogenic therapies have been shown to correlate with survival.
Pharmacokinetic/pharmacodynamic markers: Erlotinib is cleared rapidly by smokers with 25-40% lower exposure and hence cautiously increasing the dose in active smokers Journal of Carcinogenesis A peer reviewed journal in the field of Carcinogenesis and Carcinoprevention is recommended in the package insert. Exposure to imatinib was assessed in a study of 73 patients who were randomly assigned to 400 or 600 mg of imatinib daily for advanced GIST, there was a 10-fold variance in trough levels with either dose (from 414 to 4182 ng/mL). [103] Clinical outcomes were correlated with trough levels at steady state. Trough values below 1100 ng/mL correlated strongly with a significantly shorter time to tumor progression and a lower rate of clinical benefit as compared to higher trough levels and authors have suggested lower exposure may contribute to drug resistance.

CONCLUSION
The "one size fits all' approach for cancer therapy has long gone. With integration of optimum technology and better understanding of how cancers evolve at the molecular level, newer potential therapeutic targets are discovered faster than ever before. This understanding has helped us to embrace personalized approaches to treat cancer. Since the approval of bevacizumab in 2004 for colon cancer, there are now nine approved targeted drugs for treatment of GI cancers (five for colon, two for PNET, one for gastric/gastroesophageal, one for HCC, one for pancreas) and small incremental benefits have been seen in survival in each of these cancers. The era of personalized medicine is here, identifying validated assays and targets and doing studies in selected populations where the effect size is large as the population is preselected with patients who are most likely to benefit, has set a new standard for developing novel targeted agents in GI cancers. As their survival is generally poor and cost of drugs and toxicity is high, reducing sample size of trials and focusing on clinically meaningful rather than merely statistically significant benefit has become our driving principle for the future.
The results of ongoing and planned trials in colon cancer seek to expand the current approved indications for these agents, since the successful approval of bevacizumab as second line after failure of bevacizumab containing first line regimen. Notable phase 3 trials that are ongoing or planned include using ziv-aflibercept in the first line setting and adjuvant trials post metastatectomy that include regorafenib. Other studies plan to evaluate integration of targeted therapies with liver directed therapies, e.g. FOXFIRE global study evaluating incorporation of sirspheres with FOLFOX bevacizumab versus FOLFOX bevacizumab alone in advanced colon cancer and sorafenib with or without chemoembolization. The RADIANT-4 trial that seeks to re-examine the role of everolimus in carcinoids has completed accrual and is expected to be analyzed at the end of 2014. Given the success of trastuzumab in gastric cancer, the analysis of the LOGIC trial that has completed accrual and examines the role of adding lapatinib to standard chemotherapy in HER-2 positive gastric cancer is eagerly awaited. With the exception of erlotinib, all tyrosine kinase inhibitors approved for treatment of GI malignancies have been approved as single agents. More trials evaluating the benefits of targeted therapy in the adjuvant setting for GI cancers are needed, with better risk assessment to aid decision making as is used now for imatinib in the adjuvant setting for GIST tumors. The STORM study evaluating the value of adjuvant sorafenib 400 mg BID in delaying/preventing recurrence after radio frequency ablation or surgery for potentially curable HCC has completed accrual and results are awaited. Several ongoing trials of chemotherapy plus a tyrosine kinase inhibitor are ongoing or planned and despite many notable failures of this approach especially in pancreatic cancer, many investigators remain optimistic that the preclinical success with this approach may translate into clinical benefit. Biomarker driven approaches are ideal, but limited availability of tissue for such studies and lack of validated assays has been a major challenge. As a class of agents combined VEGF and FGFR inhibitors appear promising, but as yet have not received approval for any malignancy. In addition, many trials of vaccines and immune modulatory agents are planned or ongoing and integration with chemo and targeted therapies is also of great interest.


extfieldsofstudy: Biology Medicine

text:
The biosynthetic pathway for a thousand-year-old natural food colorant and citrinin in Penicillium marneffei

Monascorubrin and its derivatives are polyketides used as natural colorants for a wide range of food for more than one thousand years. Since the biosynthetic pathway for this ancient chemical compound is unknown and genome sequence unavailable for any Monascus species, monascorubrin production has relied on extraction from fungal cultures of Monascus species. In vitro synthesis and genetic manipulation are not possible. Here we report the polyketide gene cluster and pathway for monascorubrin biosynthesis in Penicillium marneffei, a diffusible red pigment-producing, thermal dimorphic fungus, taking advantage of available genome sequence and faster growth rate than Monascus species. We also documented that the red pigment of P. marneffei is a mixture of more than 16 chemical compounds, which are amino acid conjugates of monascorubrin and rubropunctatin, and showed that this polyketide gene cluster and pathway are also responsible for biosynthesis of ankaflavin and citrinin, a mycotoxin with nephrotoxic activity in mammals. The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species.

M onascorubrin and its related compounds are polyketides that have been used as natural red colorants for a wide range of food, such as red wines, tofu and meats for more than one thousand years in China, Japan and other Southeast Asian countries 1 . The earliest known reference to use these red colorants was in a recipe for red pot-roast lamb, in which meat was simmered with hong qu (red rice koji, made with Monascus purpureus), recorded in the Qing Yilu in AD 965 2 . In Pen Ts'ao Kang Mu (Compendium of Materia Medica), the most complete and comprehensive herbal medicine book ever written in the history of traditional Chinese medicine, complied by Li Shi-zhen in the 16 th century, he described the utilization of these red pigments as a coloring agent and as a medicine in the treatment of medical diseases 3 . Subsequently, these red pigments were also used in the western world in the meat industry, for coloring sausages and hams 4 . In 2005, more than 50 patents have been issued in Japan, America, France and Germany, on the use of Monascus pigments for food; with an annual consumption of these pigments in Japan increased from 100 tons in 1981 to 600 tons by the end of the 1990s 5 . Throughout the years, production of monascorubrin and its related compounds has relied on extraction from cultures of fungi of the genus Monascus, most commonly M. purpureus. The chemical pathway for monascorubrin biosynthesis has never been elucidated in any fungal species. Moreover, no genome sequence is available for any Monascus species. Therefore, in vitro synthesis and manipulation by genetic engineering are not possible. Citrinin, another polyketide and mycotoxin with nephrotoxic activity in mammals, was found to be present in the cultures of many Monascus species, including M. purpureus 4,6,7 . Regulations have been set up to limit the content of citrinin in the pigments.
Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis [8][9][10][11][12][13] . Biologically, it is one of the most important causes of opportunistic mycosis in HIV positive patients in China and Southeast Asia. P. marneffei produces black, yellow and red pigments. In addition to its thermal dimorphic property, the production of the characteristic diffusible red pigment by its mold form is the most important characteristic for laboratory identification of P. marneffei. Recently, we have sequenced the genome of P. marneffei [14][15][16] . Interestingly, 23 putative PKS genes and two putative PKS-non-ribosomal peptide synthase hybrid genes were identified in the P. marneffei genome, a diversity much higher than those of other pathogenic thermal dimorphic fungi, such as Histoplasma capsulatum (one PKS gene) and Coccidioides immitis (10 PKS genes) 17 . We have also characterized the black and yellow pigments of P. marneffei, which are synthesized by one and two of the 25 PKS genes respectively 17,18 . Due to its relatively fast growth rate, which is twice as fast as M. purpureus, and the availability of genome sequence, it is an ideal model fungus for fungal genetic studies.
Since monascorubramine has been found in cultures of P. marneffei 6 , we hypothesized that it is the red diffusible pigment of P. marneffei and it is synthesized by one of the PKS gene clusters in its genome. To test the hypothesis, we systematically knocked down all 25 PKS genes of P. marneffei. We also knocked down genes upstream and downstream to the PKS gene responsible for red pigment production and characterized the pathway for biosynthesis of the red pigment. Uniquely, the diffusible red pigment of P. marneffei is a mixture of more than 16 chemical compounds and these compounds as well as ankaflavin and citrinin were synthesized by the same PKS gene cluster.

Results
Red pigment biosynthesis gene cluster in P. marneffei is composed of pks3 and four neighboring genes. All 25 PKS genes of P. marneffei were systematically knocked down. A loss of the red pigment was observed exclusively in the mold form of the pks3 knockdown mutant (Fig. 1), which has a pks3 transcription level , 1% of that in wild type. The pks3 gene is 8104 bp in length. It has one intron of 52 bp (from 619 to 670 bp). The resultant putative mRNA encodes 2684 amino acid residues with a predicted molecular mass of 293.68 kDa. PKS3 has one ketosynthase, one acyltransferase, two acyl carrier proteins, one methyltransferase and one thiolester reductase domains (Fig. 1). This domain organization is consistent with clade III multi-aromatic ring PKS among fungal non-reducing PKSs under Kroken et al. classification 19 .
Five ORFs upstream (orf1 to orf5) and nine ORFs downstream (rp1 to rp4 and orf6 to orf10) to pks3 were systematically knocked down (Fig. 1). RT-PCR confirmed that the 14 ORFs were successfully knocked down ( Supplementary Fig. S1). A loss of the red pigment was observed in the mold form of four of the knockdown mutants (Fig. 1). These four ORFs (rp1 to rp4) were immediately downstream to pks3, encoding putative transcriptional activator, fatty synthase beta subunit, 3-oxoacyl-[acyl-carrier-protein] synthase and oxidor- The domain structure of pks3 is shown. (b) Each knockdown mutant was grown on Sabouraud dextrose agar after 7 days incubation at 25uC. The degrees of the silencing transcriptional levels of the respective genes in knockdown mutants are indicated in brackets. ACP, acyl carrier protein; AT, acyltransferase; KS, ketosynthase; MT, methyltransferase; R, thioester reductase.
By UV-Vis spectroscopic analysis, four absorption maxima, 373, 405, 499 and 553 nm, were recognized in the culture medium growing the mold form of wild type P. marneffei (Supplementary Table  S2). The absorption maximum at 499 nm, which indicated the presence of red color, was not observed in the culture medium growing the mold form of the pks3, rp1, rp2, rp3 and rp4 knockdown mutants and the RPMI medium control. Furthermore, the absorption maxima at 373 nm and 405 nm, which indicated the presence of yellow color, were found in the wild type and rp2, rp3 and rp4 knockdown mutants but not observed in the culture medium growing pks3 and rp1 knockdown mutants. The results were in line with the color observed when the strains were cultured on Sabouraud dextrose agar (Fig. 1). For the other ten knockdown mutants (orf1-orf10), the measured absorption maxima were the same as that of wild type P. marneffei.
Red pigment of P. marneffei is composed of monascorubrin, rubropunctatin and their amino acid conjugates. To elucidate the identity of the red pigment, ultra-high performance liquid chromatography (UHPLC) profiles of wild type and pks3, rp1, rp2, rp3 and rp4 knockdown mutants of P. marneffei were monitored using DAD at scan range from 200 nm to 640 nm. Peaks that were present in the wild type but not in pks3, rp1, rp2, rp3 and rp4 knockdown mutants were recorded and subjected to mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. All the MS and MS/MS data were adjusted to mass accuracies within 10 ppm and 20 ppm respectively. At retention time 20.6 min, a peak corresponding to a compound with m/z 405.1672 and UV absorption maximum at 475 nm was observed in the wild type, but not in pks3, rp1, rp2, rp3 and rp4 knockdown mutants in positive ionization mode of the UHPLC-MS analysis ( Fig. 2a and b). The molecular formula of this compound, C 23 H 26 O 5 Na, matched that of the sodium adduct of monascorubrin. The protonated monascorubrin ion, with m/z 383.1865, was selected for further confirmation of its identity by MS/MS analysis operated in positive ionization mode (Fig. 2c) 1 . Another fragment ion, butane ion, gave the peak at m/z 57.0703. All the UHPLC-MS and MS/MS data matched those using the monascorubrin standard, confirming that this compound observed in the red pigment was monascorubrin.
In addition to monascorubrin, peaks were also observed in wild type but not pks3, rp1, rp2, rp3 and rp4 knockdown mutants of P. marneffei at retention time 16 23.197 and 23.209 min. Using UHPLC-MS and MS/MS, these peaks were identified to be the arginine, lysine, asparagine, serine, glutamic acid, tyrosine, glycine, aspartic acid, tryptophan, valine, methionine, leucine, isoleucine and phenylalaine conjugates of monascorubrin respectively (Supplementary Fig. S2-S15 and  Supplementary Table S3). Notably, the peak at 18.901 min represented two amino acid (asparagine and serine) conjugates of monascorubrin. Furthermore, peaks were observed at 13.407 and 19.730 min. Using UHPLC-MS and MS/MS, these peaks were identified to be the arginine and phenylalaine conjugates of rubropunctatin (the molecular formula of which is that of monascorubrin minus C 2 H 4 ) respectively (Supplementary Fig. S16-S17 and Supplementary Table S3).
To verify that the red pigment of P. marneffei is composed of monascorubrin, its amino acid conjugates and the amino acid conjugates of rubropunctatin, wild type P. marneffei was cultured in Aspergillus minimal medium supplemented with each of the 20 essential amino acids. The colors of the cultures were identical to those of monascorubrin or rubropunctatin conjugated with the corresponding amino acids (Fig. 3).
Red pigment biosynthesis pathway in P. marneffei is also responsible for ankaflavin and citrinin biosynthesis. To determine whether the red pigment biosynthesis pathway in P. marneffei is also responsible for synthesis of other polyketides, additional peaks that were present in wild type but not in some or all of the pks3, rp1, rp2, rp3 and rp4 knockdown mutants of P. marneffei were searched for.
At retention time 18.9 min, a peak corresponding to a compound with m/z 387.2166 and UV absorption maxima at 285 nm, 390 nm and 510 nm was observed in wild type, but not in pks3, rp1, rp2, rp3 and rp4 knockdown mutants in positive ionization mode of the UHPLC-MS analysis ( Fig. 2d and e). The molecular formula of this compound, C 23 H 30 O 5 , was compatible with ankaflavin. The identity was confirmed by MS/MS analysis (Fig. 2f).
At retention time 12.8 min, a peak corresponding to a compound with m/z 251.0914 and UV absorption maxima at 240 nm, 282 nm and 333 nm was observed in wild type and rp2, rp3 and rp4 knockdown mutants but not in pks3 and rp1 knockdown mutants in positive ionization mode of the UHPLC-MS analysis ( Fig. 2g and h). The molecular formula of this compound, C 13 H 14 O 5 , was compatible with citrinin. The identity was confirmed by MS/MS analysis (Fig. 2i). All the UHPLC-MS and MS/MS data matched those using the citrinin standard, confirming that this compound observed was citrinin.
Intermediates of the pathway for red pigment, ankaflavin and citrinin biosynthesis in P. marneffei. To elucidate the biochemical pathway for red pigment, ankaflavin and citrinin biosynthesis in P. marneffei, intermediates accumulated in the pks3, rp1, rp2, rp3 and rp4 knockdown mutants were searched for.
At retention time 9.4 min, a peak corresponding to a compound with m/z 233.0808 and UV absorption maxima at 240 nm and 338 nm, implying that the compound was yellow in color, was observed in wild type and rp2, rp3 and rp4 knockdown mutants but not in pks3 and rp1 knockdown mutants in positive ionization mode of the UHPLC-MS analysis ( Fig. 4a and b). This compound, corresponding to protonated compound 1, with the molecular formula C 13 H 12 O 4 was observed. The identity was confirmed by MS/MS analysis (Fig. 4c) and rp4 knockdown mutants as compared to wild type as its levels were 2.8 to 6.4 folds higher in rp2, rp3 and rp4 knockdown mutants than in wild type (Fig. 4a). At retention time 8.2 min, a peak corresponding to a compound with m/z 185.1183 and UV absorption maxima at 400 nm and 485 nm was observed in pks3 and rp1 knockdown mutants, but not in wild type and rp2, rp3 and rp4 knockdown mutants in negative ionization mode of the UHPLC-MS analysis (Fig. 4d and e). The molecular formula of this compound was compatible with the deprotonated fatty acid 3-oxo-decanoic acid, C 10 H 18 O 3 . The identity was confirmed by MS/MS analysis (Fig. 4f)

Discussion
Through generating a series of targeted gene knockdown mutants, we identified the genes encoding the hypothetical biosynthesis pathway for the red pigment in P. marneffei (Fig. 5). The pathway involves a gene cluster of five genes, including pks3, rp1, rp2, rp3 and rp4. The red pigment of P. marneffei is composed of at least 16 compounds when cultured in RPMI 1640 medium, including monascorubrin and its arginine, lysine, asparagine, serine, glutamic acid, tyrosine, glycine, aspartic acid, tryptophan, valine, methionine, leucine, isoleucine and phenylalaine amino acid conjugates and rubropunctatin and its arginine and phenylalaine amino acid conjugates that we detected in the present study. These results and speculations are in line with the findings of experiments that showed a perfect match between the colors of the medium culturing P. marneffei with the presence of only one amino acid and those of monascorubrin and rubropunctatin conjugated with the same amino acid (Fig. 3). We infer that the amino acids were added to monascorubrin and rubropunctatin, resulting in Schiff base formation, after generation of monascorubrin and rubropunctatin. This is in line with experiments that showed that amino acids can be conjugated to monascorubrin and rubropunctatin under specific conditions without enzymatic catalysis 20 . The conclusions drawn on the identity of the red pigment in the present study is in contrast to that of a previous article by Bhardwaj et al., which claimed that the red pigment of P. marneffei structually resembled herquinone and appeared as a dimer through disulfide bond 21 . In the present study, such a compound was not observed in wild type P. marneffei and the knockdown mutants.
PKS3 is responsible for catalyzing multiple steps on the biosynthesis pathway, whereas RP2, RP3 and RP4 catalyze the formation of 3-oxo-decanoic acid, and RP1 is the transcriptional activator of PKS3 (Fig. 5). Since RP1 is a predicted transcriptional activator and the metabolomic profile of rp1 knockdown mutant was identical to that of pks3 knockdown mutant, RP1 should be the transcriptional activator of pks3. Since rp2, rp3 and rp4 knockdown mutants shared the same metabolomic profile, their gene products should catalyze the same part of the red pigment biosynthetic pathway. In fact, RP2 and RP3 are predicted subunits of fatty acid synthase with very high amino acid identities to the subunits of fatty acid synthase of Talaromyces stipitatus, the teleomorph of Penicillium emmonsii. Therefore, RP2 and RP3, together with RP4 should catalyze the synthesis of 3-oxo-decanoic acid, which was observed in pks3 knockdown mutant because compound 1, the other substrate for 3-oxodecanoic acid to react with and form monascorubrin, was not available. When rp2, rp3 or rp4 was knocked down, compound 1 was accumulated to a level much higher than that of wild type PM1. On the other hand, compound 1 was not detected in pks3 and rp1 knockdown mutants. These indicate that PKS3 catalyzes the synthesis of compound 1 and the utilization of compound 1 is catalyzed by RP2, RP3 and RP4. Specifically, the addition of a methyl group at C5 of the precursor of compound 1 should be catalyzed by the methyltransferase domain of PKS3. Both 3-oxo-decanoic acid and a polyketide chromophore were postulated intermediates of monascorubrine biosynthesis in M. purpureus, although the polyketide The red pigment biosynthetic pathway in P. marneffei is also responsible for biosynthesis of ankaflavin and citrinin. It has been speculated that monascorubrin and ankaflavin are synthesized by the same biochemical pathway in M. purpureus due to their structural resemblance 23 . In this study, we showed that both monascorubrin and ankaflavin were not detectable in all the pks3, rp1, rp2, rp3 and rp4 knockdown mutants. Therefore, it is logical to deduce that monascorubrin and ankaflavin are both end products of this biochemical pathway, where ankaflavin is the reduced product of monascorubrin. As for citrinin, it was not detectable in pks3 and rp1 knockdown mutants, but detectable in rp2, rp3 and rp4 knockdown mutants. We infer that citrinin is an early side product of the pathway (Fig. 5). This is in contrast to M. purpureus, where it has been shown that monascorubrin and citrinin are synthesized by two separate pathways because when the PKS gene responsible for synthesis of citrinin was disrupted, red pigment production from the fungus was not affected 4 . We speculate that the yellow pigment observed in rp2, rp3 and rp4 knockdown mutants is a mixture of citrinin and compound 1.
The present study on elucidation of the biosynthetic pathway of monascorubrin is a proof-of-the-concept study that serves as a cornerstone for future studies on monascorubrin biosynthesis pathway dissection in Monascus species. Carbon-13 labeling experiment in M. purpureus has shown that monascorubrin was synthesized from one acetate and five malonate molecules 24 . The only genetic information on red pigment biosynthesis in M. purpureus includes a gene and its possible transcriptional activator, named MpPKS5 and mppR1 25 . Deletion of mppR1 resulted in a loss of red pigment and monascor-ubrin 25 . Since MpPKS5 and mppR1 possess 65% and 49% amino acid identities to PKS3 and RP1 of the red pigment biosynthesis cluster in P. marneffei, it is logical to deduce that a similar cluster should also be present in M. purpureus and other Monascus species. Further studies would shed light on ex vivo manufacturing of these ancient pigments.

Methods
Strain and culture conditions. P. marneffei strain PM1 was isolated from an HIVnegative patient suffering from culture-documented penicilliosis in Hong Kong 14 . The yeast form of PM1 was used for DNA extraction for knockdown of the PKS genes and genes in the neighborhood of pks3. A single colony of the fungus grown on Sabouraud dextrose agar at 37uC was inoculated into yeast peptone broth and incubated in a shaker at 37uC for 10 days.
Knockdown of PKS genes and genes in the neighborhood of pks3. DNA extraction was performed using the DNeasy Plant Mini Kit according to manufacturer's instructions (Qiagen, Hilden, Germany). The extracted DNA was eluted in 50 ml of AE buffer and the resultant mixture was diluted 103 and 1 ml of the diluted extract was used for PCR.
Plasmid construction was performed according to our previous publication 17 . Knockdown of pks1 to pks25 was reported previously 18 . For knockdown of rp1, plasmid pSilent-1 26 , obtained from the Fungal Genetics Stock Center, was used to construct the pPW2422 plasmid. First, the internal rp1 fragment (sense) was amplified using primers LPW18206 59-CCGCTCGAGCTGCTGGCGATACCGAGTTC-39 and LPW18207 59-CCCAAGCTTGGGGCAAGGCATCAGCTCAATGA-39 (Invitrogen, USA) (Supplementary Table S4 TGCTGGCGATACCGAGTTC-39 and LPW18209 59-GAAGATCTGCAAGG-CATCAGCTCAATGA-39 (Invitrogen, USA), using the PCR conditions described above. This amplified fragment was purified as described above, digested with BglII and KpnI, and cloned into the BglII-KpnI site of the pPW2422-1, resulting in pPW1459. The wild type P. marneffei strain PM1 was transformed with linearized pPW2422, using 200 mg/ml hygromycin for selection. For the other genes in the neighborhood of pks3, they were knocked down using the protocol described above with primers listed in Supplementary Table S4. RT-PCR and real-time quantitative RT-PCR. Total RNA was extracted using RiboPure-Yeast (Ambion, USA). The RNA was eluted in 70 ml of RNase-free water and was used as the template for RT-PCR and real-time quantitative RT-PCR. Reverse transcription was performed using the SuperScript III kit (Invitrogen, USA). PCR and agarose gel electrophoresis were performed according to our previous publication 27 . Real-time RT-PCR assays was performed as described previously 17,18 , for rp1 fragment with primers LPW18206 and LPW18207 (Supplementary Table S4), using actin with primers LPW8614 59-CAYACYTTCTACAAYGARCTCC-39 and LPW8615 59-KGCVARRATRGAACCACC-39 for normalization. cDNA was amplified in a LightCycler 2.0 (Roche, Switzerland) with 20 ml reaction mixtures containing FastStart DNA Master SYBR Green I Mix reagent kit (Roche, Switzerland), 2 ml cDNA, 2 mM MgCl 2 and 0.5 mM primers at 95uC for 10 min followed by 50 cycles of 95uC for 10 s, 57uC (55uC for actin gene) for 5 s and 72uC for 23 s (36 s for actin gene). For the other genes, real-time RT-PCR was performed using the protocol described above with primers listed in Supplementary Table S4.
Sequence analysis of PKS3 and genes in the neighborhood for red pigment biosynthesis. Introns were predicted by performing pairwise alignment with the annotated Talaromyces stipitatus (teleomorph of Penicillium emmonsii) complete genome sequence. Domains of PKS3 were predicted using the Conserved Domains Database of NCBI and PFAM (http://pfam.sanger.ac.uk/search?tab5searchSequence Block) and manual inspection of multiple alignments of PKS3 and its homologous sequences.
Fermentation and extraction of red pigment. Ten-day-old yeast cultures of wild type, rp1, rp2, rp3, rp4 and pks3 knockdown mutant strains of P. marneffei were washed twice with Milli-Q water. Four hundred microliters of each culture at a turbidity of McFaland 1, and 400 ml of Milli-Q water as the negative control, were grown in 10 ml RPMI 1640 medium (Gibco) supplemented with 5% glucose at 25uC with shaking at 250 rpm for 100 h. After incubation for 100 h, 5 ml of culture medium from each strain and control were filtered with a 0.22 mm filter. Metabolic activities in the culture medium were quenched by incubating the filtrates in liquid nitrogen for 10 min. The filtrates were lyophilized for 48 h. The lyophilized samples were reconstituted in 800 ml solution of water, methanol and acetonitrile (25454) mixture and vortexed for 5 min. The samples were undergone ultrasonic extraction for 10 min, followed by centrifugation at 12000 rpm for 5 min. Supernatants were used for UV-Vis spectroscopic examination and UHPLC-DAD/ESI-Q-TOF-MS analysis.
UV-Vis spectroscopic analysis. UV-Vis spectroscopic analysis was performed according to our previous publication 18 . The maximum absorbance of the extracts was examined by a UV-Vis spectrometer (NanoDrop 1000 spectrophotometer, Thermo scientific, USA) from 300 to 750 nm. The absorbance was measured by using 0.2 mm path.
UHPLC-DAD/ESI-TOF-MS analysis. UHPLC-DAD/ESI-TOF-MS analysis was performed according to our previous publication 18 . Separations were performed using Agilent 1290 UHPLC (Agilent Technologies, USA) and Agilent Eclipse Plus RRHD C18 (2.1 3 100 mm, 1.8 mm) column with Agilent SB-C8 (2.1 3 30 mm, 3.5 um) guard column, in both positive and negative ionization modes. The injection volume was 8 ml of each sample. The column and the autosampler temperature were maintained at 50uC and 10uC respectively. For the positive mode, the separation was performed at a flow rate of 0.4 ml/min under a gradient program. Mobile phase A was 0.1% acetic acid (v/v) in water. Mobile phase B was pure methanol. The gradient program was applied as follows: t 5 0 min, 2% B; t 5 1 min, 2% B; t 5 10 min, 45% B; t 5 24 min, 99% B; t 5 27 min, 99% B; t 5 27.01 min, 2% B; t 5 30 min, 2% B. The capillary voltage was kept at 13800 V with nozzle voltage at 10 V. For the negative mode, the separation was performed at a flow rate of 0.4 ml/min under a gradient program. Mobile phase A was 5 mM ammonium acetate with 0.1% acetic acid (v/v) in water. Mobile phase B was pure methanol. The gradient program was applied as follows: t 5 0 min, 25% B; t 5 1 min, 25% B; t 5 15 min, 99% B; t 5 20 min, 99% B; t 5 20.1 min, 25% B; t 5 25.0 min, 25% B. The capillary voltage was kept at 23800 V with nozzle voltage at 20 V. Mass spectrometer was operated in both positive and negative ESI mode using Agilent 6540 A Q-TOF mass spectrometer (Agilent Technologies, USA) with Agilent Jet Stream ESI source. The gas temperature was kept at 300uC. The drying gas (nitrogen) was set at 7 L/min and the pressure of the nebulizer gas (nitrogen) was maintained at 45 psi. The sheath gas was kept at a flow www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 6728 | DOI: 10.1038/srep06728 rate of 10 L/min and was maintained at a temperature of 350uC. The voltages were kept at 130 V, 50 V and 500 V of the fragmentor, skimmer 1, and octopoleRFPeak, respectively. The full scan mass range was adjusted to 50-1700 m/z at the acquisition rate of 2 spectra/s. Data were acquired and processed using Agilent MassHunter Qualitative Analysis software (version B.03.01, Agilent Technologies, USA). Rubropunctatin (ReseaChem GmbH, Switzerland), monascorubrin (ReseaChem GmbH), octanoic acid (Sigma Aldrich), 7-oxo-octanoic acid (Sigma Aldrich) and citrinin (Sigma Aldrich) were used as authentic standards in both MS and MS/MS analysis.
Culture of P. marneffei in Aspergillus minimal medium with different amino acids. Ten-day-old yeast cultures of wild type and pks3 knockdown mutant strains of P. marneffei were washed twice with Milli-Q water. Four hundred microliters of each culture at a turbidity of McFaland 1 were grown in 10 ml Aspergillus minimal medium 28 supplemented with one of the 20 essential amino acids at a concentration of 25 mM with shaking at 25uC for 72 h at 250 rpm and observed for pigment production. The colors of the cultures were compared to monascorubrin and rubropunctatin standards conjugated with the corresponding amino acids.


s2fieldsofstudy: Biology Medicine

text:
Integrin-mediated resistance to epidermal growth factor receptor-targeted therapy: an inflammatory situation

Targeting the function of epidermal growth factor receptor (EGFR) has failed as an effective clinical option for breast cancer. Understanding the drivers of inherent resistance has been a challenge. One possible mechanism is the acquisition of stem-like properties through the process of epithelial-mesenchymal transition. A recent study by Seguin and colleagues adds to our understanding of this process by demonstrating a functional role for unligated αvβ3 integrin in mediating a stem-like phenotype and facilitating resistance to EGFR-targeted therapy via enhanced downstream coupling to a KRAS:RalB:NF-κB pathway. Importantly, the identified mechanism may reveal a possible strategy for sensitizing breast cancer cells to EGFR-targeted therapies.

Targeting the function of epidermal growth factor receptor (EGFR) has failed as an effective clinical option for breast cancer. Understanding the drivers of inherent resistance has been a challenge. One possible mechanism is the acquisition of stem-like properties through the process of epithelial-mesenchymal transition. A recent study by Seguin and colleagues adds to our understanding of this process by demonstrating a functional role for unligated αvβ3 integrin in mediating a stem-like phenotype and facilitating resistance to EGFR-targeted therapy via enhanced downstream coupling to a KRAS:RalB:NF-κB pathway. Importantly, the identified mechanism may reveal a possible strategy for sensitizing breast cancer cells to EGFR-targeted therapies.

Background
Expression of epidermal growth factor receptor (EGFR) serves as a robust marker for the basal-like subtype of breast cancer (BC) and a biomarker for decreased survival times of BC patients [1,2]. Moreover, numerous studies have established functional roles for epidermal growth factor/EGFR in facilitating the systemic dissemination of BC cells [3][4][5]. However, clinical trials targeting EGFR for the treatment of BC have produced disappointing results [6,7]. These contrasting findings suggest that while BC cells utilize EGFR signaling to facilitate certain aspects of tumor progression, they become inherently resistant to inhibition of EGFR function during the later stages of disease progression. Defining the drivers of this type of inherent resistance has been difficult because, as opposed to acquired resistance, these cells are resistant prior to therapeutic intervention.
Integrin receptors are vital conduits that mediate cellcell and cell-extracellular matrix interactions, as well as initiating intracellular signaling cascades promoting survival and migration. The complexity of signaling events that emanate from these receptors has prevented effective targeting of their function for the treatment of BC. β3 integrin is a well established marker of highly aggressive and metastatic BC [8]. Furthermore, aberrant expression of β3 integrin is strongly related to epithelialmesenchymal transition [9], a process involved in several aspects of tumor metastasis [10], generation of a stemlike state [11] and resistance to EGFR-targeted therapies [12]. However, the functional role of β3 integrin in mediating a stem-like state and resistance to EGFR-targeted therapies has remained undefined.

The article
In a recent article published in Nature Cell Biology, Seguin and colleagues [13] demonstrate the importance of an αvβ3:KRAS:RalB:NF-κB signaling pathway in driving a stem-like state and resistance to EGFR-targeted therapies. In this work, the authors first demonstrate that patientderived tumor tissues can be enriched in tumor-initiating potential based on expression of β3 integrin. Correspondingly, depletion of β3 integrin in tumor cell lines decreases stem-like cells as measured by several assays. Importantly, use of the nonadherent tumor sphere assay was the first indication that the role of β3 integrin in modulating tumor initiating capacity was independent of its role in cell adhesion. Next, the authors demonstrate that β3 integrin is necessary and sufficient to impart growth resistance to the EGFR inhibitor erlotinib. Interestingly, expression of β3 integrin had no impact on the response to nucleoside analogues or DNA crosslinking drugs, suggesting this result is restricted to targeted therapies. Moving downstream of β3 integrin the authors expand upon their previous findings [14] and show that the ability of β3 integrin to facilitate drug resistance does not function through the more traditional focal adhesion complex, but instead via a KRAS:RalB: TBK1:NF-κB pathway that is facilitated by galectin-3. Finally, the authors demonstrate that combining erlotinib with the proteasome inhibitor bortezomib can overcome β3 integrin-mediated resistance to EGFR inhibition (EGFRi).

Viewpoint
With the enhanced specificity and reduced toxicity of targeted molecular therapies comes the drawback of inherent and acquired resistance. As stated above, several lines of evidence point to EGFRi as an effective therapeutic strategy for BC, but these predications have not been borne out in the clinic. Together with previous findings, the current study suggests that targeting of galectin-3, RalB [15] or TBK1 [16] may actually be capable of inducing sensitivity to EGFRi therapies. Of particular importance is the treatment of triple-negative BCs, which currently lack any effective targeted therapeutic option. With regard to the other BC subtypes, whether this β3:NF-κB mechanism of resistance takes place during acquisition of resistance to HER2-targeted or endocrine-targeted therapies also remains to be determined [17]. Similar to EGFRi, vascular endothelial growth factor (VEGF)-targeted therapy has only had limited success in BC treatment. Several clinical trials currently underway are combining the VEGF-targeted agent bevacizumab with bortezomib, but further investigations into the role of β3 integrin:NF-κB in this resistance process are clearly warranted.
Mechanistically, the finding that β3 integrin induces stemness during the unligated state is very intriguing. Indeed, cilengitide is a cyclic peptide antagonist of αv binding that is currently in clinical trials for the treatment of multiple cancers. However, the current study demonstrates that this compound is unable to target the stem-like cells that drive drug resistance, suggesting it may not be an effective therapy for BC. Specificity remains a major drawback to compounds targeting the NF-κB pathway. Despite this, several ongoing clinical trials are exploring the combination of bortezomib with erlotinib and other targeted therapies [18]. Using β3 integrin as a histological marker, these clinical approaches can be evaluated for eradication of the stem-like cell population in the hopes of unlocking the potential of erlotinib as an effective BC therapy.


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Cell-Cell Adhesions and Cell Contractility Are Upregulated upon Desmosome Disruption

Desmosomes are perturbed in a number of disease states – including genetic disorders, autoimmune and bacterial diseases. Here, we report unexpected changes in other cell-cell adhesion structures upon loss of desmosome function. We found that perturbation of desmosomes by either loss of the core desmosomal protein desmoplakin or treatment with pathogenic anti-desmoglein 3 (Dsg3) antibodies resulted in changes in adherens junctions consistent with increased tension. The total amount of myosin IIA was increased in desmoplakin-null epidermis, and myosin IIA became highly localized to cell contacts in both desmoplakin-null and anti-Dsg3-treated mouse keratinocytes. Inhibition of myosin II activity reversed the changes to adherens junctions seen upon desmosome disruption. The increased cortical myosin IIA promoted epithelial sheet fragility, as myosin IIA-null cells were less susceptible to disruption by anti-Dsg3 antibodies. In addition to the changes in adherens junctions, we found a significant increase in the expression of a number of claudin genes, which encode for transmembrane components of the tight junction that provide barrier function. These data demonstrate that desmosome disruption results in extensive transcriptional and posttranslational changes that alter the activity of other cell adhesion structures.

Introduction
Desmosomes are robust cell-cell adhesion structures that provide mechanical integrity to tissues. They are particularly abundant in the epidermis and heart due to the significant mechanical stresses these tissues experience. The importance of desmosomes is highlighted by the many diseases associated with mutations in desmosomal genes. Depending on the gene affected and the severity of the mutation, symptoms can range from focal skin thickening and curly hair to lethal blistering disorders [1]. In addition, the autoimmune diseases pemphigus vulgaris (PV) and pemphigus foliaceus result from pathogenic antibodies against desmosomal cadherins [2,3]. Finally, bacterial toxins target desmosomes in Staphylococcal scalded skin syndrome [4].
The response of cells and tissues to desmosome disruption is complex. In pemphigus, a number of studies have identified signaling pathways that become activated by pathogenic antibodies [5][6][7][8][9]. In addition, microarray analyses have revealed significant changes in transcript profiles upon treatment of human keratinocytes with PV sera [10]. However, previous studies have not examined in detail whether desmosome disruption affects adherens junction or tight junction activity, the other two prominent cell-cell adhesion structures in the epidermis. Furthermore, we do not know whether the responses to genetic disruption of desmosomes are similar to those elicited by autoimmue disruption.
In addition to desmosomes, both cultured keratinocytes and the epidermis have robust adherens junctions and tight junctions. Adherens junctions are structurally similar to desmosomes, and some studies have demonstrated a requirement for adherens junctions in desmosome and tight junction formation [11][12][13][14][15]. In addition, loss of desmoplakin resulted in changes in the morphology of both adherens junctions and the actin cytoskeleton in cultured keratinocytes [16]. However, the functional status of adherens junctions was not further investigated. In contrast, overexpression of the head domain of desmoplakin in A431 cell resulted in desmosome defects, but no overt adherens junction defects were reported [17]. While desmosomes and adherens junctions provide mechanical integrity and adhesion strength to the epidermis, tight junctions are essential for the barrier function of the skin [18]. Tight junction function has not been examined upon disruption of desmosomes.
In addition to their role in binding keratins, we previously reported that desmosomes are required for microtubule reorganization during epidermal differentiation. The reorganization of microtubules to the cell cortex has two important effects: 1) it strengthens adherens junctions through forces generated by myosin II, 2) it increases tight junction barrier activity [19]. Because of these previous findings, we wanted to determine whether loss of desmosomes resulted in changes in the composition, expression or function of these other important cell adhesion structures. Alterations in these could either exacerbate or ameliorate the effects of loss of desmosomes, and thus, may be important diagnostically and/or therapeutically.

Myosin II-dependent changes in adherens junctions in desmoplakin-null cells
To better understand the cellular and tissue responses to loss of desmosomes, we began by examining the status of adherens junctions in desmoplakin-null mouse keratinocytes. We found that both the transmembrane protein E-cadherin and the peripheral membrane protein a-catenin were localized to cell-cell junctions in both wild type and desmoplakin-null keratinocytes grown in the presence of 1.2 mM calcium for 24 hours (Fig 1A,B). Although adherens junction components were present at cell-cell contacts, the organization of those contacts was distinct in the desmoplakinnull cells. Rather than having junctions that formed relatively straight lines, the localization of adherens junction proteins in desmoplakin-null cells was more punctate, reminiscent of their localization during adherens junction assembly. This phenotype was seen in three independently derived DP null keratinocyte lines. The adherens junction phenotype in these cell lines was similar to, but somewhat less dramatic, than that previously reported in primary mouse keratinocytes [16]. In addition, localization of E-cadherin and other adherens junction proteins was normal in intact desmoplakin null epidermis [16]. Thus, loss of desmoplakin does not result in a significant impairment of the localization of adherens junction components to cell-cell contacts, although it may affect either their maturation and/or their morphology.
Our previous data demonstrated that desmosomes are essential for microtubule reorganization in keratinocytes [19,20]. The reorganization of microtubules to the cell cortex resulted in changes in adherens junctions that were consistent with them being under tension. One marker that we and others have used to examine the tension status of the adherens junction is the a18 antibody [19,21]. This antibody recognizes an epitope of a-catenin that is not accessible under low-tension conditions, but becomes exposed when the adherens junction is under tension [21]. Additionally, exposure of this epitope is lost upon myosin II inhibition, further suggesting that this is due to tension [19,21]. That said, it is important to note that this antibody recognizes a conformation of a-catenin that could alternatively be induced independent of tension. As one marker of adherens junction status, we therefore examined the exposure of the a18 epitope in desmoplakin null cells. To our surprise, we found that this epitope was always exposed in desmoplakin-null cells, in contrast to its induced exposure by increasing tension (through taxol treatment) in wild type cells (Fig 1 C-E). Quantitation of the relative cortical/ cytoplasmic intensity demonstrated that the enrichment of a18 at the desmoplakin-null cell cortex was significantly higher than the wild type control, but lower than the wild type cells treated with taxol. As expected, taxol treatment did not cause a further increase in cortical a18 levels in desmoplakin null cells. This is consistent with our previously published work demonstrating that desmoplakin null cells cannot reorganize their microtubule to the cell cortex [19,20]. To determine whether the exposure of the a18 epitope was indeed myosin II-dependent in this context, we treated DPnull cells with the myosin II inhibitor blebbistatin. Inhibition of myosin II resulted in a decrease in cortical staining with the a18 antibody, indicating that myosin II-dependent forces act on adherens junctions in the absence of functional desmosomes (Fig 1E, F). Blebbistatin treatment did not demonstrably alter the localization of E-cadherin or a-catenin, so the effects are unlikely to be secondary to junctional reorganization (Fig 1I, J). These data suggest that DP-null cells have a higher degree of junctional contractility at steady state compared to WT cells. WT and DP-null keratinocytes stained for E-cadherin (green) and a-catenin (red) after 24 h in Ca 2+ . (C-F) WT (C,D) and DP-null (E,F) keratinocytes stained with the tension-sensitive anti-a-catenin antibody, a18 (red). The a18 epitope is exposed in WT keratinocytes after microtubules are reorganized to the cell cortex with taxol treatment, 1 hour at 10 mM. (D). DP-null keratinocytes have the a18 epitope exposed at steady state (E). The junctional intensity is not significantly changed upon treatment with taxol (F). (G) Quantitation of junctional/cytoplasmic intensity of a18 in indicated samples. n.100 cells from at least two independent experiments. p values as compared to WT DMSO are ,0.0001 for both WT with taxol and DP KO DMSO. There was no significant difference between DP KO DMSO verses taxol. Myosin II is upregulated in desmoplakin-null epidermis To understand this change in adherens junction status, we began by examining the levels and localization of type II myosins in intact epidermis and in cultured cells. In the WT epidermis, myosin IIA levels were low at embryonic day (e) 17.5 and increased by e18.5 (Fig 2A, C). In contrast, myosin IIA was easily detectable at e17.5 and was present at elevated levels at e18.5 in DP cKO epidermis (Fig 2B, D). This was especially evident in the suprabasal epidermis where desmosomes are most abundant. Western blot analysis of epidermal lysates confirmed an increase in the total amount of myosin IIA in the mutant epidermis at e18.5 ( Fig 2E). This change was not specific to myosin IIA, as both myosins IIB and C were present at higher levels in the DP cKO epidermis ( Fig 2E). We next examined myosin IIA localization in cultured WT and DP-null keratinocytes to get a higher resolution view of its subcellular organization. We found an approximately 2fold increase in the levels of cortical myosin IIA in DP-null cells compared to WT cells (Fig 2F-H), which is consistent with increased cortical contractility and increased tension on adherens junctions.

Treatment with pathogenic pemphigus antibodies results in increased recruitment of myosin II to the cortex
The changes in myosin IIA levels and localization could either be due to specific loss of desmoplakin or a general response to desmosome perturbation. To test this, we examined the effects of desmosome perturbation by treatment with the monoclonal pathogenic anti-Dsg3 antibody, AK23 [22]. Because treatment times were relatively short, we examined the localization but not the total levels of myosin IIA. Similar to the loss of desmoplakin, AK23 treatment resulted in increased association of myosin IIA with cell junctions (Fig 3A-C). This closely resembled the myosin IIA localization in DP-null cells or in WT cells with cortical microtubules. However, it was not due to loss of cortical desmoplakin, as, consistent with a previous report [23], we found no significant effect of AK23 treatment on desmoplakin localization (Fig 3G-H). Therefore, perturbation of desmosomes by two distinct methods resulted in changes in myosin II localization.
The increased association of myosin II with cell-cell junctions upon AK23 treatment was particularly interesting, as it suggested that contractility may play a role in the disease pathology of pemphigus. However, the presence of myosin II at the cortex of AK23-treated cells does not necessarily mean that adherens junctions were altered. We therefore examined whether the a18 epitope of a-catenin was exposed upon AK23 treatment. Staining with the a18 antibody revealed robust cortical exposure of the epitope after AK23 treatment (Fig 3D-F), demonstrating a change in adherens junctions' status upon pathogenic antibody treatment, similar to the effects of genetic loss of desmoplakin.

Myosin IIA exacerbates pemphigus-induced tissue fragility
Increased myosin II contractility and engagement of adherens junctions could result in one of two disparate outcomes. First, this could be a protective effect -by upregulating adherens junction engagement and adhesion, cells could compensate for the loss of desmosome-mediated adhesion. This would suggest that loss of myosin contractility would exacerbate the effect of pemphigus antibodies, resulting in increased tissue fragility. Alternatively, in the absence of robust desmosomes, the increase in tension provided by myosin II contractility could promote cell separations by physically pulling cells apart from one another. Thus, the loss of myosin II would prevent or decrease fragility induced by pemphigus antibodies. To distinguish between these possibilities, we treated WT and myosin IIA-null keratinocytes with control or pathogenic pemphigus antibodies. Mutant cells showed no defect in cell sheet strength under control conditions, as assayed by dispase-based cell sheet disruption assays. However, while AK23 antibodies caused an increase in the fragility of WT keratinocytes, myosin IIA-null keratinocytes were protected from AK23-induced fragility ( Fig 3I). These data suggest that myosin IIA activity promotes tissue fragility in response to desmosome disruption. However, it is also possible that myosin II works in a noncanonical fashion to promote cell separations.

Tight junction changes upon desmosome disruption
In cultured keratinocytes, the tension-induced engagement of adherens junctions leads to an increase in tight junction-dependent barrier activity [19]. To determine whether this pathway was functional in DP-null cells, we began by examining the localization of two tight junction proteins in a calcium shift assay in which differentiation and cell adhesion are initiated by the addition of 1.2 mM calcium to the medium. Two interesting differences between the WT and DP-null keratinocytes were noted. First, there was a delay in ZO-1 localization to cell-cell contacts in the DP-null cells (Fig 4 A-D). This was noted in all three cell lines generated. However, by 8 hours after calcium addition, the localization of ZO-1 was similar to the WT controls (Fig 4A9-B9). Early ZO-1 localization to the cell cortex is likely due to its association with adherens junctions [24,25], and we noted colocalization between E-cadherin and ZO-1 at these early time points (Fig 4E). While ZO-1 localized to cell-cell adhesions early, the transmembrane protein occludin was only sparsely found at cell-cell adhesions at early time points (Fig 4A). In WT cells, it became more broadly localized to cell junctions by 24 hours after calcium addition. In contrast, there was a clear increase in the kinetics of the association of occludin with the membrane in DPnull cells, and junctions were uniformly labeled by 16 hours after calcium addition compared to 24 hours in WT cells (Fig 4B-B9). Quantitation at 16 hours revealed that 94+/24% of WT ZO1 junctions were occludin positive, while only 44+/29% of KO (3 independent experiments, n.100 for each).

Loss of desmoplakin results in transcriptional changes of claudins
Tight junction are regulated in various ways, including the expression levels of distinct claudins, transmembrane components of the tight junction that are necessary for tight junction strand formation [26]. We began by examining the mRNA levels of various claudins that have previously been reported to be expressed in the epidermis under normal or pathogenic conditions. We found significant increases in three of the eight claudin genes we examined -Claudins 1 (9X), 6 (6X) and 12 (4X) (Fig 4F). Not only was claudin-1 mRNA increased in DP-null cells, but the level of the protein was also significantly higher in mutant versus wild type control lysates (Fig 4G). These changes in claudin-1 levels were found, to varying degrees, in the two additional independently generated desmoplakin null cell lines (Fig 4H). Therefore, an increase in claudin-1 levels was a reproducible effect of loss of desmoplakin. Immunofluorescence analysis demonstrated that desmoplakin null keratinocytes had increased intracellular vesicular pools of claudin-1 as compared to the wild type controls (Fig 4J,K).
When we tested tight junction function by measuring transepithelial resistance, we found that different desmoplakin null keratinocyte lines had significantly different TER levels. While two cell lines had TER values near wild-type levels (250 Vcm 2 ) one desmoplakin null line had TER approximately 10X wild-type levels (2500 Vcm 2 ). Therefore, loss of desmoplakin is not inconsistent with functional TER, however, this occurs in the absence of mechanical force. It is important to note that desmoplakin null epidermis is severely disrupted and therefore clearly does not maintain barrier function with intact tight junctions. This is likely due to mechanical stresses in intact epidermis that are absent in cultured cells.
We next wanted to determine whether the changes in levels of claudin-1 also occurred in vivo, to determine whether this might be a marker for desmosome dysfunction in intact tissue. By immunofluorescence analysis, we noted an increase in intensity of claudin-1 staining (Fig 4L,M), which we confirmed by western blot analysis of lysates prepared from WT and DP-null suprabasal epidermis (Fig 4I). Therefore, both in vitro and in vivo, desmoplakin loss resulted in increased levels of claudin-1.

Discussion
Desmosomes are the principle structure in the epidermis that protects the tissue from mechanical stresses. Our data have shown that perturbation of desmosomes results in robust changes in other cell-cell junctions in the epidermis. In addition, desmosome disruption results in increased myosin II levels/cortical localization that may increase junctional contractility and exacerbate blistering disease phenotypes. As all these experiments were conducted with mouse tissues and cells, it will be important to determine whether similar changes occur upon disruption of desmosomes in humans.
These studies were initiated because of our earlier findings on the role of desmosomes in promoting microtubule reorganization, and the effect that microtubules have on adherens and tight junctions [19,20]. Contrary to our expectations, we did not find loss of myosin II localization and/or adherens and tight junction function in desmoplakin null cells. Rather, both the establishment of cortical microtubules (which is desmosome-dependent) and the loss of desmosomes resulted in some similar effects, though the underlying mechanisms are clearly distinct. This suggests that robust pathways control cortical contractility and cell adhesion in both physiological and pathological situations.
Adherens junctions are known mechanosensors and mechanotransducers, able to change activity and composition under tension [21,[27][28][29]. These changes require myosin II-mediated contractility [21,30,31]. We expected that loss of desmosomal adhesion would result in loss of tension across adherens junctions in keratinocytes, as desmosomes provide the majority of the strength to resist mechanical stress. Surprisingly, we report evidence for an increase in contractility and adherens junction engagement upon desmosome disruption. This data was based, in part, upon the exposure of the a18 epitope of a-catenin. While this has been a useful surrogate for adherens junction tension, it may also reflect non-tension based changes in adherens junctions. Therefore, future biophysical approaches will be required to determine the functional status of these junctions. This is complicated by the concurrent changes in other adhesion structures. The changes in adherens junctions were associated with an increase in myosin II protein levels and increased cortical localization of myosin II, presumably due to signaling pathways activated in the absence of functional desmosomes. We propose that forces generated by the increased levels of cortical myosin II can promote cell-cell separations upon desmosome disruption. This suggests an active cellular involvement in cell separations as well as passive effects from external forces. Complete desmosome disruption by loss of desmoplakin is clearly sufficient to induce fragility. However, we propose that increased cell fragility under conditions where desmosomes are partially perturbed is promoted by myosin II function. At very low levels of perturbation it is possible that myosin II contractility actually aids cell adhesion, by promoting adherens junction engagement. However, when desmosomes are not strong enough to withstand the increased forces, this would result in overt cell separations. That said, it is important to note that the a18 antibody used in these studies reports on a conformational change of a-catenin. While this change is induced by tension, we cannot rule out that non-tension based pathways can result in similar conformational changes.
Our data also demonstrated an increase in the expression of a number of tight junction components in desmoplakin null keratinocytes, especially claudin-1. Desmoplakin null epidermis showed a similar increase in claudin-1 levels. While there have not been reports of tight junction activity in pemphigus patients, it is noteworthy that an upregulation of claudin 1 was found in Hailey-Hailey disease [32]. Hailey-Hailey disease results in blistering of the epidermis and is caused by a mutation in the ATP2C1 gene, resulting in desmosomal defects. This supports the idea that the upregulation of claudins may be a general response to loss of desmosome adhesion. Whether this is a response to increase myosin II and tension, or whether these changes are downstream of other signals is not yet known. In addition, the role these changes play in disease pathology is unclear. It is unlikely that claudin upregulation upon severe desmosome disruption is able to promote barrier formation, as the epidermis is so mechanically fragile. However, in less severe cases, this may increase barrier function to compensate for integrity defects. It is also of note that claudin-1 is present throughout the suprabasal epidermis, not just where functional tight junctions form. It is therefore possible that tight junction-independent functions for claudin-1, including promoting adhesion, also occur. However, at least in cultured DP null keratinocytes, not all of the claudin-1 makes it to cell junctions, and an increase in cytoplasmic/vesicular pools is observed. While genetic loss of function studies have demonstrated an essential role for claudin-1 in the epidermis [18], similar studies have not been performed for claudins 6 and 12. Surprisingly, overexpression of claudin 6 alone in differentiated epidermis resulted in differentiation and barrier defects, demonstrating that claudins can have unexpected roles on epidermal function [33]. Importantly, cultured desmoplakin knockout cells did not have defects in transepithelial resistance, suggesting that in the absence of mechanical forces, desmosomes are not necessary for this function of tight junctions.
One of the most interesting questions raised by this study is the nature of what cells and tissues are sensing when desmosomes are disrupted. Clearly, a number of signaling pathways are activated upon treatment with pemphigus sera, but it is not clear whether any of these also respond to genetic disruption of desmosomes. In theory, the cell could sense changes in signal transduction pathways controlled by desmosomes, the organization of the cytoskeleton that is altered upon desmosome disruption, or cell-cell adhesion through contractile mechanisms. Whether there is any mechanical sensing and signaling in response to pemphigus antibodies is an important question that will require further investigation. In this way, myosin II may be important for both sensing defects in adhesion as well as participating in the effects of desmosome disruption. Finally, whether these changes that we have identified or the signaling pathways that lead to them can be clinically targeted to ameliorate the effects of desmosome perturbation is an important outstanding question.

Cell culture and Mouse Studies
All studies were performed on mouse tissue and mouse keratinocytes. All animal studies were performed with approval from the Duke Animal Use and Care Program. Keratinocytes were isolated from the backskin of e18.5 embryos by dispase treatment and trypsinization. After several passages on fibroblast feeders, keratinocyte lines were grown in the absence of feeders in E low calcium media (3:1 DMEM:F12 (Invitrogen) with insulin (Sigma, 0.5 mg/ml), cholera toxin (ICN Biomedicals, 0.1 nM), transferrin (Sigma, 0.5 mg/ml), hydrocortisone (Calbiochem, 0.4 mg/ml) and T3 (Sigma, 0.2 mM) with 15% FBS (Hyclone), which was chelated in Chelex (BioRad) to remove calcium. Calcium was supplemented to 0.05 mM with CaCl 2 . Three independent DP null keratinocyte lines were developed. At least two different lines were used for each experiment, and in some cases all three (as stated in text). For experiments, keratinocytes were grown to confluency and calcium was added to 1.2 mM to induce adhesion. For drug experiments, 24 hours after calcium switch, cells were treated with 10 mM blebbistatin (Sigma, an inhibitor of myosin II) or 10 mM taxol (Sigma, a microtubule stabilizer) for 1 hour. Cells were treated with 2 mg anti-Dsg3 antibodies (AK23, Medical and Biological Laboratories) for 4 hours before examination (added to cells with intact junctions). AK23 is a pathogenic mouse-anti-Dsg3 antibody [34,35].
Keratinocytes were grown on glass coverslips in E low calcium media until confluent. Cells were induced to differentiate and form junctions by the addition of calcium to 1.2 mM. Twenty-four hours after addition of calcium, cells were fixed in either 4% paraformaldehyde in PBS +0.2% Triton X-100 for 8 minutes or in -20uC methanol for 2 minutes. They were blocked with 3% BSA, 5% NGS, and 5% NDS in PBS+0.2% Triton X-100 for 15 minutes. Primary antibody was incubated for 15 minutes, and secondary antibody was incubated for 10 minutes. Coverslips were mounted in a solution of 90% glycerol in PBS with 2.5 mg/ml p-Phenylenediamine (Sigma-Aldrich). Samples were imaged on a Zeiss AxioImager Z1 microscope with Apotome attachment and MRm camera. Junction/cytoplasm intensity was quantified in ImageJ. For each image two random regions of the cytoplasm were used to determine background fluorescence levels. Cortical fluorescence was determined by taking the mean of the fluorescence intensity over the cell junction and subtracting the background (cytoplasmic) levels.

Western Blotting
The backskin of e18.5 mice was incubated with Dispase II (1.2 U/ml in PBS) overnight at 4uC to isolate the epidermis. The epidermis was minced and boiled in Laemmli sample buffer.
TER TER was measured as previously reported [19]. Briefly, cells were grown to confluence on Transwell inserts (Corning), calcium was added to 1.2 mM and transepithelial resistance was measured 48 hours after calcium addition with a MilliCell ERS-2 V-ohmmeter (EMD Millipore).

Cell sheet integrity assays
Cell sheet integrity was measured as previously reported [19]. Briefly, mouse keratinocytes were grown in 6-well dishes to confluency, then calcium was added to 1.2 mM. 24 h after calcium addition, cells were treated with 4 mg antibody for 4 h, then dispase II (1.2 U/ml in PBS) was added to lift the cells off of the dish. The cell sheets were subjected to mechanical disruption by pipetting up and down 10 times. The number of sheet fragments was counted manually.

Statistical analysis
Statistical analyses were performed in GraphPad Prism software. A student's t-test was used to determine statistical significance. P,0.05 was considered statistically significant.


s2fieldsofstudy: Biology Chemistry

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A Review of Novel Combinations of Colistin and Lipopeptide or Glycopeptide Antibiotics for the Treatment of Multidrug-Resistant Acinetobacter baumannii

As antibiotic resistance continues to increase among Gram-negative organisms such as Acinetobacter baumannii there is a growing need for novel therapies to overcome these resistance mechanisms. Antibiotics active against multidrug-resistant A. baumannii (MDRAB) are few, and agents in development are primarily active against other multidrug-resistant Gram-negative organisms. The combinations of colistin and antimicrobials such as glycopeptides and lipopeptides are unique potential treatment modalities against MDRAB. For both lipopeptides and glycopeptides in vitro data have demonstrated significant synergy, resulting in rapid bactericidal activity in time-kill curves. Several invertebrate in vivo models have also demonstrated increased survival compared to colistin alone. Currently, very little clinical data have focused on using these combinations for infections caused exclusively by multidrug-resistant Gram-negatives. The combination of vancomycin and colistin has been studied with conflicting results regarding both improved outcomes and risk of nephrotoxicity. Although in vitro and in vivo models have proved promising, further investigation is required to provide clinical data necessary to support the use of these combinations. The objective of this review is to summarize literature currently available for the novel combination of lipopeptides or glycopeptides with colistin for the treatment of A. baumannii, in particular MDRAB. Electronic supplementary material The online version of this article (doi:10.1007/s40121-014-0051-9) contains supplementary material, which is available to authorized users.

INTRODUCTION
Antibiotic resistance continues to be a global public health crisis [1]. The Centers for Disease Control and Prevention (CDC) state that resistant bacteria cause over two million infections in the United States annually, with a mortality rate of over 20,000 individuals per year [2,3]. Curtailing resistance through infection control measures is key to mitigating the threat and decreasing the spread of these organisms [4]. Once patients are infected, however, optimizing antimicrobial therapy becomes paramount. This is a challenge secondary to the declining availability of active agents and dwindling pipeline of novel  [19][20][21][22]. This review will focus on several unconventional combinations with colistin that have demonstrated possible clinical utility.

METHODS
Literature associated with the combination of colistin and glycopeptides, lipopeptides, and lipoglycopeptides for the targeted treated of ACB was included in this review. All relevant English-language peer-reviewed publications were accessed through PUBMED using the following medical subject heading (MeSH) terms: vancomycin, daptomycin, telavancin, glycopeptide, lipopeptide, lipoglycopeptide, colistin, Acinetobacter baumannii, and combination therapy. Citations within these references were also assessed for inclusion. Publications in abstract form were included in the review if these abstracts were presented as part of professional meetings.
The analysis in this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors.

Studies with Glycopeptide Antibiotics
Until recently little was known about combinations of colistin and antimicrobials with exclusive activity against Gram-positive bacteria. Novel combinations of colistin plus glycopeptide and lipopeptide antibiotics have recently been examined, yielding positive results [23,24]. Vancomycin is a glycopeptide antibiotic that has become a mainstay in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) over the past two decades that works by inhibiting peptidoglycan synthesis in the bacterial cell wall [25,26]. The proposed mechanism of synergy relates to the cellmembrane permeabilizing ability of colistin.
Colistin causes an electrostatic interaction with LPS (lipid A), disrupting the outer membrane of the Gram-negative bacteria. This allows the otherwise large and hydrophobic vancomycin molecules to pass through the outer LPS layer and reach the site of action at the cell wall [23,24].
Synergy between vancomycin and colistin in MDRAB was first described by Gordon  Gram-positive co-infection (41.2% versus 0.0%, p\0.001). Thirty-day mortality was not significantly different between those treated with the combination and those treated with monotherapy (33.8% versus 29.6%). Although infection with MDRAB was an independent predictor of mortality, through cox regression the receipt of the combination for C5 days was shown to be protective for 30-day survival.
There was no difference in renal toxicity between the patients who received glycopeptides and those that did not.

Studies with Lipopeptide Antibiotics
In addition to the glycopeptides, the lipopeptide antibiotic daptomycin and lipoglycopeptide antibiotic telavancin have been investigated in combination with colistin (Table 1). Telavancin has a dual mechanism of action wherein it acts as glycopeptide antibiotics by inhibiting transpeptidation and transglycosylation, the peptidoglycan polymerization step of cell wall synthesis, as well as altering cell membrane permeability via changing membrane potential [35,36]. Similar to vancomycin and teicoplanin, the combination with colistin has demonstrated significant synergy in vitro and improved survival in an in vivo invertebrate model [30,37]. Daptomycin has a unique and not yet fully elucidated mechanism of action that causes calcium-dependent destabilization of the cell membrane as well as altering the cell wall through septal defects [38]. As is the case with glycopeptides, the presence of a LPS outer membrane in Gram-negative bacteria protects against daptomycin binding to the cellular membrane.  isolates that were exposed to 0.5 9 MIC colistin bactericidal activity occurred in five (50%), compared to zero isolates exposed without daptomycin. Nine of 10 isolates exposed to colistin at 1 9 MIC demonstrated bactericidal activity with no regrowth at 24 h. It appears the possible benefits of the daptomycin-colistin  impact of these combinations is required to determine the possible benefits associated with use of such combinations. In particular, elucidation of the mechanism by which these combinations seem to work best in ACB as opposed to other MDR Gram-negative pathogens and further investigation of whether higher doses of daptomycin will improve results in MDRAB [24,40,41].
Additionally, further description of use of these combinations in monomicrobial MDRAB infections without de-escalation to colistin monotherapy following eradication of coinfecting Gram-positive pathogens would be beneficial from a clinical perspective. It is also unclear if utilizing polymyxin B in lieu of colistin would provide similar synergistic activity against ACB or other MDR Gramnegative pathogens. Lastly, triple therapy with colistin, a lipopeptide or glycopeptide, and another agent active against MDRAB such as tigecycline or carbapenems should also be investigated in the clinical setting. With a dwindling pipeline of agents with novel mechanisms of action, finding new and unique means to employ antibiotics that are currently available is crucial to improve outcomes in these life-threatening infections.