extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Expression of Tight Junction Protein Claudin-1 in Human Crescentic Glomerulonephritis

The origin of crescent forming cells in human glomerulonephritis (GN) remains unknown. Some animal studies demonstrated that parietal epithelial cells of Bowman's capsule (PECs) were the main component of proliferating cells and PEC-specific tight junction protein claudin-1 was expressed in crescentic lesions. We investigated the expression of claudin-1 in human GN. Immunohistochemistry for claudin-1 was performed on 17 kidney biopsy samples with crescent formation. Colocalization of claudin-1 with intracellular tight junction protein ZO-1 was also evaluated by immunofluorescence double staining. Claudin-1 is expressed mainly at the cell to cell contact site of proliferating cells in cellular crescentic lesions in patients with these forms of human GN. Small numbers of crescent forming cells showed extrajunctional localization of claudin-1. Colocalization of claudin-1 with ZO-1 was found at cell to cell contact sites of adjacent proliferating cells. In control samples, staining of claudin-1 was positive in PECs, but not in podocytes. Our findings suggest that claudin-1 contributes to crescent formation as a component of the tight junction protein complex that includes ZO-1. Co-localization of claudin-1 with ZO-1 implies the formation of functional tight junction complexes in crescentic lesions to prevent the interstitial damage caused by penetration of filtered molecules from Bowman's space.

Introduction
Extracapillary proliferative cellular lesions or cellular crescents are a hallmark of severe inflammatory reactions in glomeruli and have been documented in various forms of glomerulonephritis (GN) [1]. Crescents are observed in various forms of GN including rapidly progressive GN, postinfectious GN, IgA nephropathy, purpura nephritis, and systemic lupus erythematosus. Though the origin of these crescentic lesions remains controversial, recent studies have clarified that not only inflammatory cells, but also intrinsic glomerular epithelial cells (i.e., parietal epithelial cells of Bowman's capsule (PECs) and glomerular epithelial cells or podocytes) contribute to the development of these crescents [2]. Smeets et al. demonstrated, using the genetic cell lineage tracing method, that PECs constitute the principal component of cellular crescents, especially at the early stage, in animal models of crescentic GN [3].
Claudin is a tetraspan-transmembrane protein and is a crucial component of tight junction complexes in epithelial and endothelial cells [4,5]. Among the claudin subtypes, claudin-1 is expressed in the tight junctions of PECs in both murine models and human patients with GN and is therefore regarded as a marker of PECs [3,6,7].  Considering these observations, it is probable that PECs are the principal component of cellular crescents in various forms of human GN and that claudin-1 participates in the formation of cellular crescents by forming tight junctions among proliferating cells. We aimed to investigate the expression and localization of tight-junction protein claudin-1 in crescentic lesions of human GN.

Material and Methods
2.1. Subjects. Kidney biopsy samples from 17 patients with crescent formation were studied. Four kidney biopsy samples without crescent formation (minor glomerular abnormalities, minimal change nephritic syndrome, IgA nephropathy, and membranous nephropathy) and morphologically normal portions of kidneys from 5 patients with noninvasive renal tumors, who underwent nephrectomy, were used as control samples. Among those providing biopsy samples with crescent formation, five patients had IgA nephropathy, two had purpura nephritis, seven had myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis, one had proteinase 3 (PR3-) ANCA associated vasculitis, one had anti-glomerular basement membrane (GBM) antibody associated rapidly progressive GN, and one had lupus nephritis. Kidney biopsy or nephrectomy was performed according to the routine diagnostic and therapeutic indication protocol used by Dokkyo Medical University Koshigaya Hospital. Acquired samples were either immediately fixed in formalin followed by embedding in paraffin, or snap-frozen at −80 ∘ C. Cellular crescents are defined as the presence of at least two cell layers in Bowman's space. The presence of crescents was identified by two nephrologists. Clinical features of patients included in this study are summarized in Table 1. All procedures in this study were conducted in accordance with the Declaration of Helsinki guidelines. The Institutional Review Board (IRB) of the Dokkyo Medical University Koshigaya Hospital approved this study (IRB approval number: 1206) and written informed consent was obtained from all participating patients.

Immunohistochemistry.
Kidney biopsy specimens or tissue blocks from morphologically normal portions of surgically removed kidneys were immediately fixed with formalin and embedded in paraffin. Three m tissue sections were deparaffinized and rehydrated in a graded ethanol series. Antigen retrieval was performed by autoclaving for 10 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase and Protein Block Serum-Free (Dako, Japan) was applied to prevent nonspecific protein binding. Rabbit polyclonal antibody for claudin-1 (ab15098, Abcam, Cambridge, UK) or rabbit polyclonal anti-ZO-1 (Invitrogen) was diluted in Dako REAL Antibody Diluent (Dako) and used as a primary antibody (dilution of 1 : 200). Sections were incubated with primary antibody for 1 h at room temperature. Then, the slides were washed with phosphate buffered saline (PBS) three times and the sections were incubated with Histofine simple stain MAX-PO (MULTI) secondary antibody (Nichirei Bioscience, Japan) according to manufacturer's instructions. Counterstaining with hematoxylin was performed for nuclear staining.

Immunofluorescence.
Three m cryosections were fixed in −20 ∘ C methanol for 10 min, washed with 7.4% PBS 3 times, treated with Protein Block Serum-Free for 30 min, and washed with PBS 3 times. Then, the samples were incubated with mouse monoclonal anti-claudin 1 antibody (1 : 100) (ab56417, Abcam) for 2 h at room temperature, washed with PBS 3 times, and incubated with Alexa Fluor 488 conjugated anti-mouse IgG (1 : 1000) (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min. Again, the samples were washed with PBS 3 times, treated with Protein Block Serum-Free for 30 min, washed with PBS 3 times, and incubated with rabbit polyclonal anti-ZO-1 (1 : 400) (Invitrogen) at 4 ∘ C overnight. Samples were washed with PBS 3 times, and incubated with Alexa Fluor 555 conjugated anti-rabbit IgG (1 : 1000) (Cell Signaling Technology) for 30 min. Nuclear staining was performed using DAPI. Immunostaining for occludin was also performed using rabbit polyclonal antioccludin antibody (Cat. 71-1500, Invitrogen, US) (1 : 200) and Alexa Fluor 555 conjugated anti-rabbit IgG. As a negative control, PBS was used instead of the primary antibodies. Immunofluorescence was examined with a laser scanning confocal microscope (OLYMPUS BX-51, Olympus, Tokyo, Japan).

Transmission Electron
Microscopy. Small pieces of kidney biopsy specimens (approximately 0.5 to 1 mm 3 ) were fixed in 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) overnight. The glutaraldehyde-fixed biopsy specimens were postfixed in 1% OsO 4 in 0.05 M phosphate buffer for 30 min and embedded in epoxy resin. Ultrathin sections (approximately 100 nm) were prepared by cupper grids, then stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1010, Nihon Densi, Tokyo, Japan).

Morphometric Analysis.
Whole glomeruli were photographed in each biopsied sample with crescentic GN ( = 17). The border of the glomerulus and claudin-1 positive area was traced and the percentage of total claudin-1 positive area in the glomerulus was measured using ImageJ v. 1.47 software (http://rsb.info.nih.gov/ij). 2 × 5 mm size of tissue blocks from nephrectomized kidney were used as a control ( = 5). The difference between control sample and crescentic GN was analyzed by wilcoxon rank-sum test using Stata version 13 (Stata Corporation, College Station, TX). The level of statistical significance was set at < 0.05.

Immunohistochemistry.
Claudin-1 showed a strong, sharp staining pattern along the cell membranes of adjacent PECs. In control samples, staining signals were mainly detected at cell to cell contact sites (Figures 1(a), and 1(b)). In cellular crescents, claudin-1 signals were mainly detected in the cell membranes of adjacent proliferating cells in all samples examined (Figures 2(a), 2(b), 2(c), and 2(d)). Occasionally, we observed cells to be positive for claidin-1 in the extrajunctional cell membrane and cytoplasm (Figure 2(d), arrow and arrowhead). No claudin-1 positive cells were detected in fibrous crescents or necrotizing glomeruli (Figure 2(e)). ZO-1 positive cells were also detected in cellular crescents (Figure 2(f), arrowheads).

Immunofluorescence.
Claudin-1 is expressed exclusively at cell to cell contact sites of adjacent PECs in glomeruli without crescent formation (Figure 3(a)). Signals are distinctively concentrated in a dotted pattern. ZO-1 staining was observed in the tight junction complexes of adjacent PECs and foot processes of podocytes (Figure 3(b)). Claudin-1 and ZO-1 were colocalized (Figure 3(c)). In cellular crescents, claudin-1 expressing cells were minority (Figure 3(d)) compared to ZO-1 expressing cells (Figure 3(e)), but lines that were double positive for both anti-claudin-1 and anti-ZO-1 were observed (Figure 3(f)). Occludin, another fundamental intercellular component of tight junction complex, was also expressed in crescentic lesion (Figure 3(g)).

Transmission Electron Microscopy.
Under electron microscopy, tight junction structure (close contacts between cell membranes of adjacent cells) was confirmed in crescent forming cells (Figure 4, biopsy sample from patient with IgA nephropathy).

Discussion
In the present study, we investigated the expression of claudin-1 and its colocalization with ZO-1 in various human glomerular diseases including IgA nephropathy, purpura nephritis, ANCA associated vasculitis (MPO-ANCA and PR-3 ANCA), anti-GBM-RPGN, and lupus nephritis. We obtained 3 important findings. First, claudin-1 was expressed mainly at cell to cell contact sites in proliferating cells in crescentic lesions. Second, nonjunctional mislocalization of claudin-1 was occasionally recognized in crescent forming cells, but not in PECs of control renal samples. Third, claudin-1 was colocalized with ZO-1, which is the fundamental intracellular component of tight junction complexes in epithelia [8], at the cell to cell contact sites of adjacent crescent forming cells.
Claudin-1 was initially discovered in 1998 by Furuse et al. as a 22 kD protein with four putative transmembrane domains [9]. Introduction of claudin-1 into fibroblasts leads to formation of tight junction strands in these intrinsically tight junction lacking cells [10]. Overexpression of claudin-1 in Madin-Darby canine kidney (MDCK) cells increases transepithelial electrical resistance and reduces paracellular flux, and claudin-1 is thus considered to contribute barrier function in epithelial cells [11]. Claudin-1 is crucial not only for tight junction formation, but also for intracellular entry of the hepatitis C virus [12] as a coreceptor of tetraspanin CD81 [13]. To date, at least 24 members of the claudin family, with tissue -specific distributions, have been identified in mice and humans. Expression patterns of claudin subtypes in nephron segments were precisely investigated in mice [14] and rabbits [15]. In glomeruli, claudin-1 [14,15] and claudin-5 [16] were identified as tight junction proteins in PECs and podocytes, respectively. Kirk et al. performed an immunohistochemical analysis of claudin expression in human renal cortex and found that claudin-1 was expressed in the parietal epithelium of Bowman's capsule, distal convoluted tubule, and collecting duct [7]. Smeets et al. demonstrated, using the genetic cell lineage tracing method, that, in a murine model of crescentic glomerulonephritis (induced by injection of anti-mouse nephrotoxic serum), the majority of the cell population in International Journal of Nephrology 7 crescentic lesions is derived from PECS and that these lesions are positive for claudin-1 [3]. Their findings, taken together with our results, suggest that claudin-1 contributes to tight junction formation among adjacent proliferating cells in crescentic lesions in human glomerulonephritis.
We also found that expression of claudin-1 is not restricted to the cell to cell contact sites of adjacent cells. We detected claudin-1 staining in the nonjunctional cell membrane and cytoplasm of the cells composing the cellular crescents in crescentic glomerulonephritis. In immunofluorescence study, not all claudin-1 colocalized with ZO-1. These cells expressing nonjunctional claudin-1 might be the infiltrating immune cells, since the expression of claudin and other tight junction proteins in immune cells has been reported [17]. On the other hand, it is well known that expression of claudin is not restricted to tight junction strands in epithelial cells. Claudin-5 can be found at the apical membrane of the rat podocytes of puromycin aminonucleoside (PAN) induced nephrosis [16], and claudin-1 can be found in basolateral membrane of rat epididymis [18]. Various factors, such as interleukin-1 [19], helicobacter pylori [20], and dexamethasone [21], are known to affect the expression and localization of claudin. Moreover, Dhawan et al. demonstrated high expression of claudin-1 in human colorectal cancer tissues and that nuclear and cytoplasmic mislocalization of claudin-1 was frequently seen as compared to the normal mucosa [22]. They confirmed, by immunoblotting, expression of claudin-1 to predominate in the nuclear and cytoplasmic fractions of SW480 cells (derived from primary colorectal cancer) and SW620 cells (derived from a metastatic lesion) as compared to RIE cells (derived from noncancerous rat intestinal cells). They also demonstrated, using these two cell lines from human colorectal cancer, that modulation of claudin-1 expression resulted in the phenotypic change known as epithelial to mesenchymal transition (EMT) and suggested -catenin signaling and Ecadherin expression to be possible mechanisms underlying these claudin-1 induced phenotypic changes. Mislocalization of junctional protein may participate in EMT, as Reichert et al. showed that in MDCK cells mutant ZO-1, which is composed of PDZ domains of ZO-1, localizes mainly in the cytoplasm, activates -catenin signaling, and decreases epithelial marker expression [23]. Bariety et al. revealed that EMT occurs in the early phase in human pauci-immune crescentic glomerulonephritis [24]. Considering these observations, it is possible that nonjunctional claudin exists in human glomerular crescent and plays some novel functions. It is proposed that the nonjunctional cell membrane claudins might represent a reservoir for junctional claudins [25]. However, the role of claudins in the nonjunctional cell membrane and cytoplasm remains uncertain in this study. Recently, Ohse et al. established conditionally immortalized mouse glomerular parietal epithelial cells [26]. It might be useful to use a PEC-specific cell line to further clarify the roles of extrajunctional claudin-1 and related signaling pathways in crescent formation. This issue needs to be elucidated in future study.
ZO-1 is a 225 kD intracellular protein that is an indispensable component of tight junction complexes [8]. ZO-1 directly binds to the COOH-terminus of claudin-1 through its PDZ1 domain [27]. The PDZ1 domain of claudin-1 plays a pivotal role in the formation of normal tight junction complexes and in exerting selective molecular permeability, that is, barrier function [28]. In normal glomeruli, ZO-1 is expressed in the slit-diaphragms of podocytes [29]. In this study, we demonstrated colocalization of ZO-1 and claudin-1 in adjacent proliferating cells in human glomerular crescent. Actually, intensity of ZO-1 signal was stronger than claudin-1, and thus claudin-1 was partially colocalized with ZO-1 (Figure 3(f)). Wide variety of cells including immune cells and glomerular epithelial cells (i.e., PECs and podocytes) contribute to the formation of cellular crescent. Not only PECs but also podocytes and immune cells are known to express ZO-1 [17]. Though parietal epithelial cells that were positive in claudin-1 have been demonstrated to be the predominant population of the cellular crescent in the murine model of crescentic glomerulonephritis [3], it is unclear whether this is true in cellular crescent in human disease. ZO-1 positive and claudin-1 negative cells in human glomerular crescents could be the infiltrating immune cells or podocytes. The minority of claudin-1 positive cells in human glomerular crescent in our study might indicate that contribution of PECs to the formation of crescentic lesion is relatively small compared to murine models of crescentic GN. Ohse et al. proposed using low and high molecular weight tracers in experimental models of anti-GBM glomerulonephritis to show not only the basement membrane in Bowman's capsule, but also the tight junctions in PECs to possibly act as a second glomerular barrier preventing interstitial damage caused by penetration of filtered molecules from Bowman's space [30]. Thus, it is possible that formation of tight junction complexes with claudin-1 and ZO-1 in extracapillary proliferating lesions contributes to minimizing the solute-leakage induced interstitial damage by endowing crescents with a barrier property in human glomerulonephritis. Further experiments like permeability assay using PEC-specific cell line would contribute to verifying this hypothesis.
In conclusion, our results demonstrate that claudin-1 is expressed in cellular crescents in human glomerulonephritis. Claudin-1 is predominantly distributed at cell to cell contact sites of proliferating cells in crescentic lesions. Slight but significant distributions of this molecule in extrajunctional cell membrane and cytoplasm suggest a nonjunctional role of claudin-1. Colocalization of claudin-1 with ZO-1 raises the possibility of functional tight junction complex formation in crescentic lesions aimed at the prevention of the interstitial damage by penetration of filtered molecules from Bowman's space.


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HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice

Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset.

published we may not be able to extend the revision period beyond three months.
I look forward to seeing a revised form of your manuscript as soon as possible. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): There is no immediate medical impact. However, the study contributes to the identification of potential treatment targets in SCA1. This is relevant.
Referee #1 (Remarks): Spinocerebellar ataxia type 1 (SCA1) is a CAG repeat expansion mutation neurodegenerative disorder for which so far there is no effective treatment. The expression of the mutant protein, ATXN1, causes SCA1 probably by changing the interaction with pathways relevant for alternative splicing in the nucleus and several regulators of transcription. Ito and colleagues investigate the influence of high mobility group box protein 1 (HMGB1) on the effects of mutant ataxin 1 in a mouse model of this neurodegenerative disease. They build on previously published evidence in neurons expressing mutant ataxin1 that suggested a role of HMGB1 in maintaining the balance between splicing and transcription. The main aim seems to be to examine the different nuclear functions of HMGB1 and the cytoplasmic roles with a focus on mitochondrial quality control. The authors report on the creation of a double-transgenic mouse (atxn1-154Q KI-HMGB1). They characterize the pathological phenotype and report on behavioural improvement and prolonged survival compared with the atxn1-154Q model. They then examine how HMGB1 might exert such beneficial effects. They describe corrected DNA damage repair systems and mitochondrial DNA repair. In addition, they find reduced synapse and autophagy gene expression that is improved in the double transgenic mice. Finally, they use adeno-associated virus HMBG1 gene transfer upon which nuclear and mitochondrial abnormalities and survival improve in atxn mutant mice. They conclude that targeting HMGB1 could be a viable approach for treating human spinocerebellar ataxia type 1. The manuscript is rich in interesting data. In particular, the pronounced improvement of survival of the mice, both the atxn1-154Q KI-HMGB1 double transgenic and the AAV HMGB1 infected, suggests that indeed HMGB1 confers a substantial benefit for atxn mutant mice. Thus, the manuscript adds to the literature and potentially opens new avenues for treatment in a model of SCA1. However, I got lost many times because of the sheer volume and complexity of the data. In my opinion the manuscript would benefit if it were less convoluted following on from a clear hypothesis. Maybe the authors could consider whether really all the data they present contribute to telling a convincing story. This may also help focus and streamline the discussion so it is easier for the reader to appreciate the authors' work. While the abstract gives a clear overview of the main findings I found the discussion difficult to follow at times.
Other points -The hypotheses could be stated more clearly in the introduction. They could state more clearly that they went from their previous findings in cells to evaluating the influence on phenotype and survival of HMGB1 in a SCA1 mouse model. -I understand the limitations of space. However, some of the figures, especially the supplementary figures, are so small that I needed a magnifying glass to appreciate them. Even then it was sometimes difficult to judge what was being shown. This is a pity since it may mean that I missed relevant information.
- Figure 2B. I find it very difficult to make out the immunogold signal and to reliably tell it apart from non-specific staining Referee #2 (Comments on Novelty/Model System): The experiments showing that HGMB1 overexpression improves SCA1 mouse phenotypes are convincing, however data showing that HMGB1 might act through mitochondrial DNA damage pathway are less convincing, since most conclusions are based on experiments performed using transfected HeLa cells and these experiments are not always conclusive. The team has previously published a paper on the topic (Nat Cell Biol 2007) showing that HMGB1 is altered in SCA1 models and over expression of HMGB1 in drosophia has beneficial effects. SCA1 and more generally polyQ disorders are dramatic neurodegenerative diseases with no treatments available. The proposed strategy is therefore interesting regarding medical impact.
Referee #2 (Remarks): The paper by Ito et al. investigates the effect of overexpressing HMGB1 in SCA1 mice (expressing polyQ-ataxin-1) and shows that HGMB1 overexpression using both HMGB1 transgenic mice and injection of AAV-HMGB1 vectors improves phenotypes of SCA1 mice, including motor function and survival. In addition the study describes an effect of HGMB1 on mitochondrial damage, suggesting that HMGB1 overexpression suppresses mitochondrial DNA damage induced by polyQataxin-1 in SCA1 purkinje neurons, corresponding to cell-type affected in SCA1. The team previously showed that HMGB1 interacts with ataxin-1 and Htt and soluble HMGB1/2 is reduced in neurons expressing polyQ-ataxin-1 or polyQ-Htt (corresponding to mutated proteins in SCA1 and Huntington's diseases, respectively). They further showed that compensatory expression of HMGBs ameliorates polyQ-induced pathology in primary neurons and in Drosophila polyQ models, possibly through inhibition of genotoxic stress signals or transcriptional repression (Qi et al., Nat Cell Biol, 2007). Thus the present study extends this work. Generally, whereas the experiments showing that HGMB1 overexpression improves SCA1 mouse phenotypes are convincing, data showing that HMGB1 might act through mitochondrial DNA damage pathway are less convincing, since most conclusions are based on experiments performed using transfected HeLa cells. In vivo data are missing regarding this point.
Several concerns should be addressed.
-Generally, the manuscript should be shortened and more focused. The Results and Discussion sections are too long. Informative results and discussion should be kept. The structure of the paper could also be improved. For instance the results obtained in HGMB1 transgenic mice and in mice injected with HGMB1-AAV vector could be described in parallel, which would prevent repetitions. The results are often discussed in the Results section instead of the Discussion section. The manuscript should be edited. Pages should be numbered. Several quantifications and statistics are based on a number of unspecified measurements. The number of measurement should be specified systematically, including in sup figures (for example see Fig  - Fig 4. The complete gels should be shown. Could the authors use quantitative RT-PCR to quantify more accurately their results? Could the authors exclude that changes in PCR efficiency between WT/atxn1 KI-HMGB1 and atxn1 KI do not reflect mtDNA conformational changes rather than increase in mtDNA damage, since HMGB proteins affects DNA conformation. What types of DNA damage are expected due to decreased HGMB1, SSB, DDB...? Could the authors investigate this? Is mitochondrial genome copy number different between WT, Atxn1 KI and Atxn1-HMGB1 mice? This should be checked. Regarding high throughput sequencing in Fig. 4D. How many times was the experiment replicated? If the results are based on one experiment only, could we exclude technical artifacts resulting from variations in sonication or in sample quality and preparation? The data could be presented using box plots, which would be easier to read. As presented in Fig. 4D, it's not clear that read length is shifted to the shorter fraction. What is the time of reads? Why is average and mitochondrial read length longer in atxn1 mice compared to WT or Atxn1-HMGB1 mice (Fig. 4E)? Is the mutation frequency statistically different between atxn1 mice and WT or atxn1-HMGB1 mice - Fig. 5. Why did the author use L929 cells for X-ray irradiation? What types of damage do they induce. Why did the only check for 8-OHdG (methods regarding experiment in Fig. 5D are missing)? How many times was the experiment replicated in Fig. 5B? Regarding statistical analyses, it is sufficient to keep the one-way anova test followed by post hoc test analysis (student's t test analysis should be removed). Same comments with respect to Fig. 5C.
It would be wise to compare the effect of decreased HMGB1 in atxn1 KI mice on mtDNA and nuclear DNA to evaluate respective contributions to SCA1 phenotype.
- Fig. 6A. Expression of HMGB1 and atxn1 is highly variable between transfected cells. How representative are the pictures shown? Fig6B. Many bands are detected following HGMB1 ChIP. The PCR does not appear to be very specific. alternatively could the additional bands correspond to broken fragments bound with HGMB1? Overall, the experiment is not conclusive.Is it possible to sonicate mtDNA and perform a more quantitative PCR? How many times was the experiment replicated? -Sup Fig. S4. Statistical analyses of microarray data should be corrected for multiple testing. Genes significantly deregulated after this correction should be considered. Why did the author use only PANTHER for pathway enrichment analysis (and not GO for example). Functional enrichment analyses should be re-done using genes deregulated after correction for multiple testing and additional methods (GO...).
-The part "HMGB1 rescues the expression of mito DNA repair genes " should be shortened to few sentences. In addition, rescue is not specific to mito genes: HMGB1 does not rescue all mito genes and rescue of non mito genes also occurs. Moreover, changes go in both direction (increased and decreased) and the consequence cannot be predicted. Conclusions should therefore be tone down, this part is too speculative and vague. Too much discussion in the results part (ex: "we found recently that mutant atxn1 directly interacts with Rpa1 and BRCA1 involved in DNA double strand break repair by homologous recombination. Therefore... Meanwhile.."). Fxn is not a DNA repair protein.
-Same comments regarding the next part "HMGB1 rescues synapse-related and autophagy-related gene expression". It should be shortened, more focused and less speculative or toned down. For example: "the decreased expression of these genes could underlie the decreased thickness of the molecular layer....". The authors focus on the expression of 3 genes only, thus "underlie" should be changed for "contribute". Also, levels of corresponding proteins (Cbl1, Dab1 and Tln2) could be determined. Fig. 7 and AAV. The sentence: "we investigated a novel gene therapy for SCA1 using HMGB1" should be modified. We cannot talk about therapy at that point (given side effects associated with HMGB1 overexpression: cancer, inflammation). Details of the constructs should be specified in Methods, not in results (it's not clear why they mix AAV1 and AAV3 subtypes). Is specific targeting of purkinje neurons expected. This should be commented. The injection mode should be justified in Methods. The sentence "...we used direct injection into the posterior fossa.... because of an expected side effect that we discuss in the Discussion" should be removed.
Western-blots using HMGB1 antibody could be performed to verify that the increase of HMGB1 mRNA in atxn-1 mice overexpressing HMGB1 associates with increased HMGB1 protein. Fig. 7K. The quality of the experiment is not optimal. How did the authors quantified data from this experiment?
-Experiments aimed to verify inflammation in mice overexpressing HGMB1 could be improved, especially since the authors claim that the strategy could be used for gene therapy. Is it possible to dose inflammatory molecules or check expression of inflammatory genes? -There is no reference to Fig S7 in the manuscript.
-The absence of effect of HMGB1 overexpression on autophagy should be tone down, since the authors do not see autophagic vacuoles in atxn1 mice (Fig S3), nor do they see interaction between beclin-1 and HMGB1 (they should be cautious with negative results).

Referee #1 (Comments on Novelty/Model System):
There is no immediate medical impact. However, the study contributes to the identification of potential treatment targets in SCA1. This is relevant. >>> Thank you very much again for the kind suggestion to shape up the manuscript. We deleted previous Fig 6A from this version. We considered to volume down other figures, but since the second reviewer requested some additional experiments, at this moment we tried to prevent increasing the volume by this revision. Naturally, we also followed the suggestion from the first reviewer, and tried to focus and clarify the line of discussion. We deleted non-essential discussion and substantially shorten Discussion.
Other points -The hypotheses could be stated more clearly in the introduction. They could state more clearly that they went from their previous findings in cells to evaluating the influence on phenotype and survival of HMGB1 in a SCA1 mouse model.
>>> We changed the order of sentences and tried to make more focused introduction following the advice of the reviewer. >>> We enlarged font sizes and tried to make explanation easy to understand.
- Figure 2B. I find it very difficult to make out the immunogold signal and to reliably tell it apart from non-specific staining >>> Thank you very much for kind evaluation and critical suggestion. In response to the comment, we newly performed in vivo experiment, in which we observed DNA damage repair of mitochondrial genome and nuclear genome after irradiation (New Fig 6). Several concerns should be addressed.
-Generally, the manuscript should be shortened and more focused. The Results and Discussion sections are too long. Informative results and discussion should be kept. The structure of the paper could also be improved.
>>> We deleted a large volume of result and discussion sections that were non-essential.
For instance the results obtained in HGMB1 transgenic mice and in mice injected with HGMB1-AAV vector could be described in parallel, which would prevent repetitions.
>>> We appreciate this suggestion from the reviewer. Following the advice we actually tried to describe transgenic data and viral vector data in parallel. However, we noticed that it made the structure of figure more complex because pathological and mechanistic analyses were based on KI and Tg mice, while final output of this paper, suggestion for a possible therapeutics, is made with viral vector. Therefore, after carefully considering the two forms of manuscript, we decided to follow mostly the previous way of presentation. However, we tried our best to delete unnecessary repetitions following the advice of the reviewer.
The results are often discussed in the Results section instead of the Discussion section. The manuscript should be edited.
>>> Following the advice, we moved these discussions in "Results" to the last paragraph of page 19 in "Discussion" (page 19-20).
Pages should be numbered.
>>> We added page numbers. Fig 5B, C, D). Fig.   -Fig. 1. The authors should specify how many HMGB1 transgenic mouse lines they have generated and used in the study. It seems they only used one. Do HMGB1 transgenic mice present some phenotypes?

>>> We systematically revealed the numbers in Fig and Sup
>>> It is exactly true that we only used one line. HMGB1 Tg mice did not show any phenotype in behavioural and pathological examinations.
- Fig 2B. Controls are missing to show the staining is specific. What cells are shown in Fig. 2B? (purkinje neurons?). How did they quantify in Fig. S3B? Did the authors also perform IHF to show HGMB1 is in mitochondria of purkinje neurons? What tissues or cells were used in Fig. 2D?

>>> We added control in Sup Fig S4C. Previous presentation was inappropriate because we showed non-Purkinje cells, and in this revision we showed only Purkinje cells in left upper panel of Fig 2B.
In Sup Fig S3B (new Sup Fig 4E), we counted all mitochondria in five Purkinje cells and calculated the ratios. In Fig 2D, whole brain tissues were used (written in the figure).

-Fig 3D. SDH and COX (mistake: CCO instead of COX in legend figure?) stainings are not too convincing (mild effects, not quantitated).
>>> We showed quantification graphs. We would not go into additional experiments, because it is the basic science issue beyond the scope of this work, and additional experiments will elongate this paper longer and more complex, which had been already extremely long and difficult to follow (Editor, would you please give us advice about it?).

Is mitochondrial genome copy number different between WT, Atxn1 KI and Atxn1-HMGB1 mice?
This should be checked.
>>> We performed Southern blot of mitochondrial genome (Fig 7G), which beautifully showed the decrease of intact mitochondrial genome. While the total amount of signals including the DNA smear are relatively near.
Regarding high throughput sequencing in Fig. 4D. How many times was the experiment replicated? If the results are based on one experiment only, could we exclude technical artifacts resulting from variations in sonication or in sample quality and preparation?
>>> The experiment was repeated with three independently different samples.
The data could be presented using box plots, which would be easier to read. As presented in Fig.  4D, it's not clear that read length is shifted to the shorter fraction.
>>> Following the advice, we changed the presentation in Fig 4D.

What is the time of reads?
>>> Time of reads is the same as read count. We changed it in this version. In sequencing with ion PGM, multiple steps affect the read count. Experimental read counts are smaller than expected number by the company, the read counts become smaller when mapped to nuclear and mitochondria genome database (large mutations might be excluded in this step, if it occurs), and the read counts become further smaller by quality check. So the most important parameters in Fig 4E are average read length and mutation ratio.
Why is average and mitochondrial read length longer in atxn1 mice compared to WT or Atxn1-HMGB1 mice (Fig. 4E)?
>>> Average length of read is shorter in KI mice. On the other hand, "mitochondrial read length" does not have to be shorter. The description "mitochondrial read length" was confusing, and it should be changed to "total read length" (which is integrated read length of multiple short fragments). Total read length is dependent on multiple factors. For sequencing with Ion PGM, after preparation of DNA, adaptor sequence was attached. Adaptor-linked DNA was amplified by PCR in emulsion to several million copies, and then they were used for ion PGM sequencing. Therefore, shorter "average length of read" could be longer "total read length".

Is the mutation frequency statistically different between atxn1 mice and WT or atxn1-HMGB1 mice
>>> We showed the statistical difference in mutation frequency in Fig 4E. - Fig. 5. Why did the author use L929 cells for X-ray irradiation? What types of damage do they induce. Why did the only check for 8-OHdG (methods regarding experiment in Fig. 5D are missing)?
>>> As you know, IR induces direct and indirect DNA damages. To confirm the extents of DNA damage in three transfection groups were similar, the 8-OHdG quantification, which evaluates indirect DNA damage sensitively, was performed. Regarding the usage of L929 cells, we tried several lines of cells including Hela cells to amplify a longer DNA fragment (10kb) for DNA damage assay and we constantly succeeded only in L929 cells. The types of DNA damage evaluated by the DNA damage assay was mostly DDSB, which is the final outcome of direct and indirect DNA damages.
How many times was the experiment replicated in Fig. 5B? Regarding statistical analyses, it is sufficient to keep the one-way anova test followed by post hoc test analysis (student's t test analysis should be removed). Same comments with respect to Fig. 5C.
It would be wise to compare the effect of decreased HMGB1 in atxn1 KI mice on mtDNA and nuclear DNA to evaluate respective contributions to SCA1 phenotype.
>>> We performed the comparison experiments with in vivo IR (Fig 6).

Expression of HMGB1 and atxn1 is highly variable between transfected cells. How representative are the pictures shown?
>>> We agree the quality of this experiment was not high. Since we are requested to delete nonessential data from this paper by the first reviewer, we decided not to use Fig. 6A.
Fig6B. Many bands are detected following HGMB1 ChIP. The PCR does not appear to be very specific. alternatively could the additional bands correspond to broken fragments bound with HGMB1? Overall, the experiment is not conclusive. Is it possible to sonicate mtDNA and perform a more quantitative PCR? How many times was the experiment replicated?
>>> We repeated the experiment and obtained much better result (new Fig 6A middle panel).
-Sup Fig. S4. Statistical analyses of microarray data should be corrected for multiple testing. Genes significantly deregulated after this correction should be considered. Sup Fig 4, we performed multiple testing (Tukey-Kramer test) and changed the numbers in Venn Gram, although we believe the suggestion is too automatic from the aspect of statistics. We evaluated the %recovery of changed genes, and for the purpose two groups comparison is suitable. We presented both cases in Sup Fig 4B and C. We deleted the lower list in Sup Fig 4C because it was redundant with Sup Table 2.

>>> In
Why did the author use only PANTHER for pathway enrichment analysis (and not GO for example). Functional enrichment analyses should be re-done using genes deregulated after correction for multiple testing and additional methods (GO...).
>>> We performed GO analysis and the conclusion was basically the same.
-The part "HMGB1 rescues the expression of mito DNA repair genes " should be shortened to few sentences. In addition, rescue is not specific to mito genes: HMGB1 does not rescue all mito genes and rescue of non mito genes also occurs. Moreover, changes go in both direction (increased and decreased) and the consequence cannot be predicted.
Conclusions should therefore be tone down, this part is too speculative and vague. Too much discussion in the results part (ex: "we found recently that mutant atxn1 directly interacts with Rpa1 and BRCA1 involved in DNA double strand break repair by homologous recombination. Therefore...
>>> We shortened the part as requested. The comment about non-mitochondrial repair genes is exactly right. Regarding the orientation of changes, most of the repair genes are well known to trigger DNA damage signal and to promote death in some situation (although the details of such switching functions are not exactly known). Therefore, it does have some meaning in either direction if the abnormal changes are normalized.
>>> However, taking the advice, we tone down and shorten the section substantially. We also changed the title of mitochondria repair genes from "rescue" to "improve", and changed the title of synapse-related genes from "rescue" to "affect".
-Same comments regarding the next part "HMGB1 rescues synapse-related and autophagy-related gene expression". It should be shortened, more focused and less speculative or toned down.
For example: "the decreased expression of these genes could underlie the decreased thickness of the molecular layer....". The authors focus on the expression of 3 genes only, thus "underlie" should be changed for "contribute". Also, levels of corresponding proteins (Cbl1, Dab1 and Tln2) could be determined.
>>> We substantially shortened the section, and deleted unnecessary speculations. We did not go into the protein level because this paper basically handles gene expression and not subsequent biological processes, and because another reviewer requested us to delete marginal data from this paper. Instead, we substantially tone-downed the possible causative meaning of these genes.
>>> We changed the title of synapse-related genes from "rescue" to "affect". We stated that the meaning of change in autophagy genes is unclear. Moreover, the section about synapse-related molecule in "Discussion" was also completely deleted.  >>> The details of construction of AAV vector had been described in Method section "AAV vector construction" of previous version. There have been technical efforts to version up AAV vectors by switching the components, which actually led to better vectors. We commented about relatively specific expression in the Method.
The injection mode should be justified in Methods. The sentence "...we used direct injection into the posterior fossa.... because of an expected side effect that we discuss in the Discussion" should be removed.
>>> We removed the sentence as recommended.
Western-blots using HMGB1 antibody could be performed to verify that the increase of HMGB1 mRNA in atxn-1 mice overexpressing HMGB1 associates with increased HMGB1 protein.
>>> We added the data in new Fig 7E.   Fig. 7K. The quality of the experiment is not optimal. How did the authors quantified data from this experiment?
>>> We performed the experiment again, obtained better data and quantified ( Fig 7K).
-Experiments aimed to verify inflammation in mice overexpressing HGMB1 could be improved, especially since the authors claim that the strategy could be used for gene therapy. Is it possible to dose inflammatory molecules or check expression of inflammatory genes?
>>> We performed qPCR for inflammatory genes with cerebellar tissues, and showed the results in new Sup Fig 2B. -There is no reference to Fig S7 in  -The absence of effect of HMGB1 overexpression on autophagy should be tone down, since the authors do not see autophagic vacuoles in atxn1 mice (Fig S3), nor do they see interaction between beclin-1 and HMGB1 (they should be cautious with negative results).
>>> We toned down as suggested (page 8, last paragraph). In Discussion, we also referred the recover of the two autophagy genes in microarray analysis suggesting a possible involvement of autophagy (page 20). Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: Please carefully address the last set of comments from both referees. As you will see, they both recommend a thorough proof-reading, shortening sentences and the results section (ref.1) and overall improve the structure of the paper. In addition, please make sure that all figures and sub panels are called for in the results section, not in the discussion. Please submit your revised manuscript within two weeks. I look forward to seeing a revised form of your manuscript as soon as possible.
***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System): The immediate medical impact is low. However, the results of this study may contribute to identifying targets for treatment of SCA1. This is relevant.
Referee #1 (Remarks): In response to this reviewer's comments the authors have improved their manuscript. The introduction now states the aims of this paper more clearly, and the discussion has been pruned. However, I still think that the results section is far too long. In addition, the structure of the manuscrupt could still be improved to make for a simpler read. The most convincing results are those of improved survival and phenotype of mice with restoration of HMBI expression. The author have added in vivo data on mtDNA damage and DNA damage to boost the HeLa cell data.
Minor points -The discussion is full of reference to figures and supplementary figures. If necessary such reference, in my opinion, should take place in the results section -There are many typos throughout the manuscript. While overall the English is acceptable, at least in some instances, the English would also benefit from some editing.
Referee #2 (Remarks): - Figure 7 is added to the new manuscript but not described in the results section. Figure 5F is also not described. Figure S3H seems to be missing.
- Figure 4A: what is the fragment around 1000bp? -p13, lines 12-16, it is unclear why there is some discrepancy between the 2 studies -p13, the paragraph starting with "We further analyzed..." could be shorten, since the results do no show functional enrichments.
-p13: sub-title should be "HMGB1 improves the expression of SOME mitochondrial genes. Same comments p14. Subtitle should be :HMGB1 affects expression of SOME synapse-related genes. The effect cannot be generalized to all of mito or synapse-related genes, since these functions are not enriched in KI or in HMGB1 KI mice.

Referee #1 (Comments on Novelty/Model System):
The immediate medical impact is low. However, the results of this study may contribute to identifying targets for treatment of SCA1. This is relevant.

Referee #1 (Remarks):
In response to this reviewer's comments the authors have improved their manuscript. The introduction now states the aims of this paper more clearly, and the discussion has been pruned. However, I still think that the results section is far too long. In addition, the structure of the manuscript could still be improved to make for a simpler read.
>> Following the comment, we substantially shortened the Result section. We reduced 2 pages from Result section by compacting expressions. But we had to add a new section for Figure 7 (which was lost in the previous version, as the reviewer pointed out) and move a part from Discussion to Result section. Taken together, 1 page was reduced from Result. -There are many typos throughout the manuscript. While overall the English is acceptable, at least in some instances, the English would also benefit from some editing.
>> We corrected the typos and also received editing by a professional English editor.

Referee #2 (Remarks):
- Figure 7 is added to the new manuscript but not described in the results section.
>> We described the result of Figure 7 in Result section.
- Figure 5F is also not described. Figure S3H seems to be missing.
>> We described about Figure 5F briefly in Result section. Fig S3H has been moved to Fig S4I. - Figure 4A: what is the fragment around 1000bp?
>> We consider that it is one of the non-specific bands, and not related to the long band. However, since we obtained a better result by repeating the PCR experiment from the same samples, we replaced the figure and the source data with new ones. We upload the new Fig 4 and the new source data.
-p13, lines 12-16, it is unclear why there is some discrepancy between the 2 studies >> PATHER and GO have different genes in their categories with homologous terms. It is the primary reason. Also both methods do not retrieve absolute conclusions. Instead they just suggest preliminary expectations.
-p13, the paragraph starting with "We further analysed..." could be shorten, since the results do no show functional enrichments.
>> We changed the part following the recommendation.
-p13: sub-title should be "HMGB1 improves the expression of SOME mitochondrial genes. Same comments p14. Subtitle should be :HMGB1 affects expression of SOME synapse-related genes. The effect cannot be generalized to all of mito or synapse-related genes, since these functions are not enriched in KI or in HMGB1 KI mice.
>> We changed subtitles following the recommendation.


text:
Targeting met mediated epithelial-mesenchymal transition in the treatment of breast cancer

Mesenchymal epithelial transition factor receptor (Met) is a receptor tyrosine kinase that plays a critical role in promoting cancer cell malignant progression. Met is activated by its ligand hepatocyte growth factor (HGF). HGF-dependent Met activation plays an important role in stimulating epithelial-mesenchymal transition (EMT) in tumor cells, resulting in increased tumor cell proliferation, survival, motility, angiogenesis, invasion, and metastasis. The HGF/Met axis has thus attracted great interest as a potential target in the development of novel cancer therapies. In an effort to suppress tumor cell malignant progression, efforts have been made to develop agents capable of inhibiting inhibit Met-induced EMT, including specific Met tyrosine kinase inhibitors, HGF antagonists that interfere with HGF binding to Met, and antibodies that prevent Met activation and/or dimerization. Tocotrienols, a subgroup within the vitamin E family of compounds, display potent anticancer activity that results, at least in part, from inhibition of HGF-dependent Met activation and signaling. The present review will provide a brief summary of the increasing importance of the HGF/Met axis as an attractive target for cancer chemotherapy and the role of tocotrienols in suppressing Met activation, signaling and HGF-induced EMT in breast cancer cells. Evidence provided suggests that γ-tocotrienol therapy may afford significant benefit in the treatment of breast cancers characterized by Met dysregulation. Electronic supplementary material The online version of this article (doi:10.1186/s40169-014-0030-5) contains supplementary material, which is available to authorized users.

Introduction
Traditional cancer chemotherapeutic agents typically exhibit non-selective activity and often produce low response rates and severe toxic side effects in patients [1]. Thus, there is currently great interest in developing novel therapeutic agents that target signaling molecules involved in the growth, survival and progression of malignant cells with high specificity. The advantage of such targeted therapies is enhanced efficacy and a reduction in adverse side effects. A promising intracellular target is the receptor tyrosine kinase, mesenchymal epithelial transition factor (Met). Expressed in cells of epithelial origin, Met is activated by its ligand hepatocyte growth factor (HGF; scatter factor), which is produced by mesenchymal cells [2,3]. HGF/Met signaling is critical for epithelial-mesenchymal interactions underlying normal functions including cell migration, morphology, cell division during tissue remodeling and repair, and organogenesis during embryonic development [3,4]. However, Met is also implicated in malignant progression. Excessive or unregulated tumor cell Met activity is associated with poor patient prognosis owing to aggressive malignant phenotype typified by enhanced angiogenesis, invasion and metastasis [5][6][7][8].
HGF-dependent activation of Met leads to receptor dimerization, tyrosine autophosphorylation, and initiation of down-stream signaling, including activation of MAPK, PI3K/Akt, and STAT pathways, all of which play an important role in cancer cell proliferation and survival [7,9,10]. However, oncogenic mutations can result in Met overexpression and/or aberrant signaling that can lead to epithelial-to-mesenchymal transition (EMT) [8,11,12]. Epithelial cells that undergo EMT lose epithelial cell characteristics and acquire a mesenchymal phenotype characterized by migratory and invasive characteristics [9,12,13]. Epithelial and mesenchymal cells differ regarding function, morphology and expression of phenotype-specific protein cell markers. Epithelial cells express high levels of cytokeratin, E-cadherin, and βcatenin proteins, whereas N-cadherin, fibronectin and vimentin proteins occur at high levels in mesenchymal cells [6,8,[11][12][13][14][15][16][17][18]. Moreover, cells undergoing EMT display cancer stem cell-like properties that are associated with enhanced malignant potential and progression [19].
Tumors exhibiting rapid growth often face insufficient blood flow to meet their high oxygen demands, but are able to adapt to hypoxic conditions by altering metabolism and phenotypic functions [20]. A typical compensatory response to hypoxia is increased production of hypoxiainducible factor-1 (HIF-1), which reportedly increases HGF-dependent MET expression and signaling [21,22]. Since cancer cell invasion and metastasis are primary causes of cancer patient death, therapeutic strategies that target and suppress malignant progression may significantly improve patient survival. Thus, HGF-dependent Met activation has emerged as an attractive target for therapeutic intervention because of its role in promoting tumor cell EMT, angiogenesis, proliferation and survival [23].
Various approaches have been used to inhibit Met activation and signaling, including agents that inhibit tyrosine kinase activity (K252a, SUii274, PHA-665752, and PF2341066), neutralizing antibodies (anti-HGF antibodies) and competitive antagonists (NK4 and uncleavable HGF) that interfere with HGF binding and activation of Met, and agents that block HGF binding to Met and/or Met receptor dimerization (anti-Met antibodies) [4]. Recent investigations also show that certain natural products display potent inhibitory effects against Met activation and signaling. Tocotrienol, a rare natural form of vitamin E, is one such natural product that potently inhibits Met activation and signaling and displays potent anticancer activity at treatment doses that have minimal or no effect on normal cell function or viability [24,25]. The present review will present a brief summary of evidence supporting the HGF/Met axis as a plausible target for cancer chemotherapy, and the role of tocotrienols in suppressing Met activation, signaling and HGF-induced EMT.
Different biological effects of Met signaling are directly associated with activation of specific intracellular pathways. HGF-induced cell proliferation is mediated by activation of the MAPK cascade [35], whereas activation of the PI3K/Akt pathway promotes cell survival and prevents caspase-dependent apoptosis [9,10,36]. The ability of HGF to induce epithelial cell morphological changes and motility requires disruption of cell-to-cell adhesion and dissociation of the basal lamina from the extracellular matrix [13,37]. HGF-induced cell scattering and invasion is achieved by activation of several different signaling molecules and pathways, including PI3K, Rac and Rho [13,37]. Additionally, Met activation can stimulate vascular endothelial growth factor (VEGF) production to enhance angiogenesis [10,26]. Met forms heterodimers with other receptor tyrosine kinases including members of the epidermal growth factor (EGF) receptor family; this crosstalk between Met and other growth factor receptors permits integration of signals during the process of phenotypic change [38].
Met mutations, overexpression and/or dysregulation commonly occur in a wide variety of cancers, characteristics that are associated with promotion of cancer progression, including tumor cell proliferation, survival, motility, EMT, angiogenesis, invasion and metastasis [9,10,39]. The majority of mutations that result in constitutive activation of Met occur in the receptor's cytoplasmic tyrosine kinase domain [31], while mutations in the autoregulatory domain of the receptor allow for persistent and unattenuated responsiveness to HGF stimulation [13,35]. Overexpression of HGF can also induce oncogenic effects through over-stimulation of the non-mutated Met receptor, which can lead to enhanced tumor cell progression and malignant phenotype [10,40,41]. Enhanced HGF expression is specifically implicated as a promoter and risk factor in breast cancer [42]. Previous studies show that elevated circulating HGF levels correlate with lower survival rates and increased risk of metastasis in breast cancer patients [43,44]. Occurrence of high levels of HGF in breast cancer biopsies is associated with an aggressive malignant phenotype [5]. Studies show that Met is overexpressed in nearly 30% of all breast cancers and is a strong independent predictor of poor patient prognosis and survival [30,43]. Microarray analysis of tissue obtained from breast cancer patients shows that elevations in Met expression occur in a significant percentage of EGFR/HER2 negative tumors [30].

Inhibitors of HGF-induced Met activation and signaling
Since Met plays an important role in malignant progression, it has emerged as a practical target in cancer chemotherapy, with various strategies currently under development to inhibit Met activation and signaling. Agents that decrease receptor tyrosine kinase activity include low molecular weight molecules that block ATP binding to Met's catalytic, thereby inhibiting receptor autophosphorylation and recruitment of molecules associated with downstream signaling [4]. Several Met tyrosine kinase inhibitors, including K252a, SU11274, and PHA-665752, have been developed and studied extensively [4]. While these agents are reported to be very effective in suppressing kinase activity, their clinical usefulness is somewhat limited because they do not inhibit Met transcription and translation [45]. Subsequently, a second generation of agents developed in recent years are currently being tested in clinical trials. The use of Met tyrosine kinase inhibitors and other Met inhibitors in the treatment of human cancers has been recently reviewed in detail [46]. The inhibitory actions of these agents are summarized in Figure 1.
Agents that inhibit HGF include NK4, anti-HGF neutralizing antibodies, and an uncleavable HGF agonist. NK4 is a HGF-like ligand that binds to Met without activating the receptor [47], whereas the neutralizing anti-HGF antibodies act on various regions of the HGF molecule to prevent HGF binding to and activation of Met [48]. The uncleavable form of HGF is not biologically active, but interacts with the ligand binding site on Met to block receptor activation [49,50]. However, HGF inhibitors have also been found to have somewhat limited use because they only suppress HGF-dependent Met activation and are not effective against mutated Met receptors that are constitutively active (4).

Tocotrienol inhibition of HGF-induced Met activation and epithelial-mesenchymal transition
Vitamin E represents a family of compounds that is divided into structurally similar tocopherol and tocotrienol subgroups [51,52]. These subgroups differ as tocopherols have a saturated, whereas tocotrienols have an unsaturated phytyl chain attached to a chromane ring structure [51,52], as shown in Figure 2. However, only tocotrienols displays potent anticancer activity at treatment doses that do not affect normal cell growth or viability [53,54]. Individual isoforms (α, β, γ, and δ) of tocopherols and tocotrienols are differentiated by degree of chromane ring methylation ( Figure 2). Previous studies show that antiproliferative and apoptotic effects of tocotrienols are mediated, at least in part, by their ability to inhibit EGF receptor family member activation and signal transduction [55][56][57]. γ-Tocotrienol inhibition of mammary tumor cell growth is mediated by suppression of receptor tyrosine kinase activity of HER3/ErbB3, HER4/ErbB4, and to a lesser extent HER2/ErbB2, but not HER1/ErbB1, and attenuation of receptor downstream pathways that include MAPK, PI3K/Akt, STAT, and NFκB signaling [55][56][57]. Subsequent work demonstrated that γ-tocotrienol is also a powerful inhibitor of Figure 1 Schematic representation of the Met receptor and therapeutic strategies currently being developed to inhibit HGF-dependent Met activation and signaling in cancer. The Met receptor has an extracellular α-chain that binds HGF and a transmembrane β-chain that contains the tyrosine kinase domain and autophosphorylation sites that are essential for interacting with substrates. Activation of Met by HGF leads to receptor dimerization and recruitment of adaptor (GAB1, Grb2, Shc) and signaling (Ras/MAPK, PI3K/Akt, Src, STAT, Shp2) proteins. Downstream signaling promotes cell proliferation, altered cytoskeletal function, decreased cellular adhesion, increased cellular invasion, decreased apoptosis and enhanced DNA transcription. Anti-HGF approaches to inhibit Met signaling include anti-HGF antibodies that neutralize HGF and antagonists that block HGF binding to the Met receptor. A second approach includes the use of anti-Met antibodies that prevent HGF binding to Met or Met dimerization. Another approach is the use of specific Met tyrosine kinase inhibitors that prevent receptor second messenger signaling. Tocotrienols have also been found to be potent inhibitors of Met activation and signaling, but the exact mechanism mediating these effects are not completely understood at present. Targeting aberrant Met signaling in cancer cells can inhibit of downstream signaling pathways involved with tumor cell proliferation, motility, viability, morphology and epithelial-to-mesenchymal transition.
Studies using + SA mammary tumor cells maintained in serum-free defined media containing HGF as a mitogen show that HGF induces a dose-responsive increase in growth and corresponding increase in Met autophosphorylation, whereas combined treatment with γ-tocotrienol blocks these effects [24]. In addition, γ-tocotrienol treatment is reported to significantly reduce total Met levels in these cells. In contrast, similar treatment with growthinhibiting doses of the Met tyrosine kinase inhibitor SU11274 inhibits HGF-dependent Met autophosphorylation, but has no effect on cellular levels of Met [24]. Similarly, tumor cells transfection with Met specific siRNA significantly inhibits HGF-dependent growth [24].
Subsequent studies confirm and extend these findings in human breast cancer cell lines [25]. Results show that treatment with either γ-tocotrienol or the Met tyrosine kinase inhibitor SU11274 alone induces dose-dependent inhibition in estrogen receptor-positive MCF-7 and estrogen receptor negative MDA-MB 231 human breast cancer cells. However, combined treatment with subeffective doses (non-growth inhibiting) of γ-tocotrienol and SU11274 results in synergistic inhibition of MCF-7 and MDA-MB-231 cancer cells, while these same treatments have no effect on growth or viability of immortalized normal MCF10A human mammary epithelial cells [25]. These finding demonstrate that tocotrienol treatment alone or in combination with Met inhibitors selectively inhibits growth of breast cancer cells without affecting normal cell growth or viability. In addition, combination treatment significantly reduces Met autophosphorylation and phosphorylation (activation) of receptor substrates involved in downstream signal transduction, including STAT1, STAT5, Akt, and NFκB (26), and significantly inhibits breast cancer cell motility and migration as determined by wound healing assay [25].
HGF-dependent Met activation in transformed epithelial cells promotes expression of a malignant mammary epithelial phenotype [8]. Met-induced tumor cell progression to a malignant phenotype is a multi-step process, characterized by the loss of epithelial polarity, loss of cell to cell adhesions, degradation of basal lamina, and increased cell migration and invasion [3,4,[26][27][28][29]]. An initial step in Metinduced malignant progression is EMT (Figure 3). EMT is essential for migration of tumor cells from the site of the primary tumor, invasion into surrounding tissues and entrance into the systemic circulation [6,8,11,12,[14][15][16][17][18]. HGF-dependent activation of Met signaling shows a direct correlation with a reduction of cadherin-based adherens junctions, followed by loss of E-cadherin and cytokeratins 8/18 expression, and increased expression of mesenchymal proteins such as vimentin [6,8,11,12,[14][15][16][17][18]. Epithelial cells that undergo EMT lose epithelial cell characteristics and acquire a mesenchymal phenotype, including changes in their morphology, increased motility and invasiveness [12].
Since combined treatment with low dose of γ-tocotrienol and SU11274 has profound inhibitory effects on tumor cell growth and HGF-dependent Met activation and signaling, additional studies aimed to determine effects of these agents on breast cancer cell epithelial and mesenchymal cell marker expression. Results show that neoplastic mammary epithelial cells grown in serum-free culture medium containing HGF as a mitogen display a strong transition from epithelial to mesenchymal cell phenotype, as evidenced by low epithelial marker and high mesenchymal marker protein expression [25]. Specifically, cells display relatively low levels of the epithelial cell protein markers E-cadherin, β-catenin, cytokeratin-8, and cytokeratin-18, and a relatively high levels of the mesenchymal protein marker, vimentin [25]. These studies also show that treatment with subeffective (non-growth inhibiting) dose of γ-tocotrienol or SU11274 alone causes only a slight reduction in EMT, characterized by a slight increase in the level of some epithelial markers (cytokeratin-8 and cytokeratin-18), but little or no change in vimentin expression [25]. However, combined treatment with these subeffective doses of γ-tocotrienol and SU11274 results in reversal of EMT, as demonstrated by changes in cell marker expression including augmentation of epithelial protein markers E-cadherin, β-catenin, cytokeratin-8, and cytokeratin-18, and corresponding reduction in mesenchymal protein marker (vimentin) expression [25]. Treatment effects on epithelial and mesenchymal cell marker expression in mammary cancer cells are shown in Figure 4. Taken together, these findings provide evidence that γ-tocotrienol inhibits HGF-dependent Met activation and signaling and causes reversal of EMT. However, additional studies are required to verify that these effects obtained in vitro occur in in vivo.
Met expression is also responsive to various types of stress, particularly low oxygen tension or hypoxia [22]. Rapidly-growing solid tumors often display insufficient blood flow and oxygen deficiency within deep inner regions of the tumor. In response to hypoxic conditions, tumor cells are able to adapt by altering metabolism and phenotypic characteristics [20,58]. This compensatory response to hypoxia is mediated by increased production of hypoxia-inducible factor-1 (HIF-1), a transcription factor that promotes cell survival during hypoxic conditions [21,[59][60][61]. HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β subunits [21]. HIF-1β is constitutively expressed in most cells, whereas HIF-1α is inducible and is characteristically over-expressed in cancer cells during hypoxic conditions [21]. Overexpression of HIF-1α is also associated with elevated production of VEGF, a growth factor that promotes tumor angiogenesis, invasion and metastasis [62]. Studies show that during hypoxia, Met expression is increased through binding of HIF-1 to the Met promoter region to amplify HGF-dependent Met activation and signaling, facilitating cancer cell malignant progression [22]. Recent studies show that during hypoxic conditions, natural and semisynthetic derivatives of tocotrienols inhibit mammary tumor cell expression of HIF-1, Akt/mTOR activity and VEGF production in vitro and in vivo [63]. Although these findings suggest that tocotrienol treatment can also act to suppress the compensatory mechanisms that promote tumor growth and survival during hypoxic conditions, possibly through the inhibition of HGF/Met activation, additional studies are required to establish a direct relationship. The effects of δ-tocotrienol and its oxazine derivation on HIF-1α expression in mammary tumor cells during CoCl 2 -induced hypoxia are shown in Figure 5. can induced multiple pathways that are involved in stimulating cancer cell proliferation, survival, motility, angiogenesis, invasion and metastasis. Normal epithelial cells display a highly differentiated morphology characterized by a single layer of cells anchored by their basal lamina to the extracellular matrix. Aberrant Met activity will stimulate cell proliferation and EMT that ultimately results in changes in morphology and behavior, characteristic of a mesenchymal-like phenotype. EMT allows cancerous epithelial cells to become more mobile, invasive and metastatic in nature.

Conclusion
It has become increasingly evident during the past decade that HGF-dependent Met activation and signaling plays a major role in malignant progression and metastasis. As a result, substantial interest has focused on developing therapeutic strategies that target the HGF/Met Figure 4 Effects of γ-tocotrienol (γT 3 ) and/or the Met tyrosine kinase inhibitor, SU11274 (SU), on the expression of major epithelial and mesenchymal cellular protein markers in + SA mammary tumor cells. Cells were incubated with control or treatment media containing subeffective doses of γ-tocotrienol (2 μM) and SU11274 (3 μM) either alone or in combination containing 10 ng/ml HGF as a mitogen for 3-days. (A) Afterwards, whole cell lysates were prepared and subjected to polyacrylamide gel electrophoresis and Western blot analysis for E-cadherin, β-catenin, cytokeratin-8, cytokeratin-18, and vimentin. α-Tubulin was visualized to ensure equal sample loading in each lane. Scanning densitometric analysis was performed for each blot to visualize the relative levels of proteins. Integrated optical density of each band was normalized with their corresponding α-tubulin and control treatment bands and shown in bar graphs. Vertical bars indicate the fold-change in protein levels in various treatment groups ± SEM as compared with their respective vehicle-treated control group. *P < 0.05 as compared to their respective vehicle-treated control group. (B) Treatment effects on immunocytochemical fluorescence staining of epithelial and mesenchymal markers in + SA mammary tumor cells after a 3-day culture period. +SA cells were seeded on 4-chamber culture slides at a density of 1x10 5 cells/chamber (3 replicates/group) and allowed to attach overnight. Cells were then washed with PBS and incubated with vehicle control or treatment defined media containing 10 ng/ml HGF for 3 days in culture. At the end of treatments, cells were fixed with 4% formaldehyde/PBS and permeabilized with 0.2% triton X-100. Fixed cells were blocked and incubated with specific primary antibodies followed by incubation with Alexa Fluor 594-or 488-conjugated secondary antibodies. axis. A variety of emerging approaches are directed toward one or more of the components within this signaling pathway. Although the majority of research conducted in this area has focused on preclinical studies using cell culture and animal experimental models, a great number of therapeutic agents (onartuzumab, rilotumumab, tivantinib, and cabozantinib) developed and refined in preclinical experiments are increasingly the subject of clinical trials. Therapies that disrupt cancer cell proliferation, survival, motility, angiogenesis, invasion and metastasis will provide great benefits in the treatment and cure of cancers that display aberrant Met activity. Recent evidence suggest that γ-tocotrienol may also provide some benefit in the treatment of breast cancers that displays Met dysregulation.

Competing interests
The author confirms that this article content has no conflict of interest. There are no financial or non-financial competing interests with other people or organizations regarding the content or interpretation of the information presented in this review.

Author contribution
The corresponding author was responsible for organizing, writing and preparing the illustrations presented in this article.  HIF-1α is a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival of cancer cells during hypoxic conditions. (A) Effects of 150 μM CoCl 2 (hypoxic, but not cytotoxic dose) alone and in combination with non-growth inhibitory doses (2 μM) of δ-tocotrienol or the δ-tocotrienol oxazine derivative, 12-((R)-6,8-dimethyl-8-((3E,7E)-4,8,12-trimethyltrideca-3,7, 11-trienyl)-9,10-dihydrochromeno[5,6-e] [1,3]oxazin-2(1H,3H,8H)-yl) dodecan-1-ol), on HIF-1α levels in + SA mammary tumor cells. Non-growth inhibitory doses of δ-tocotrienol and its oxazine derivative were selected because high doses of these agents were found to initiate apoptosis and induce cancer cell death, which would confound the interpretation of the results regarding the effects of these agents on tumor cell compensatory response to CoCl 2 hypoxia. +SA cells were seeded at concentration of 1.5X10 6 in 100 mm culture dishes and allowed to attach overnight. The following day, cells were divided into groups and exposed to their respective treatments for a 24 hr incubation period. Afterwards, whole cell lysates were prepared for Western blot analysis. (B) Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding α-tubulin, as shown in the bar graphs below their respective Western blot image. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane ± SEM. #P < 0.05 compared to the vehicle-treated control group. *P < 0.05 as compared to the hypoxic group treated with CoCl 2 alone. This figure was obtained from reference 65 with permission.