p73-regulated FER1L4 lncRNA sponges the oncogenic potential of miR-1273g-3p and aids in the suppression of colorectal cancer metastasis

Summary p73 belongs to the p53 tumor suppressor family and is involved in the suppression of metastasis. However, its specific mechanism of action remains to be elucidated. Long non-coding RNAs portray a crucial role in tumor suppression. We have identified lncRNA FER1L4 as a p73 transcriptional target. The binding of p73 to FER1L4 promoter was established by bioinformatics analysis, luciferase reporter, and ChIP assays. Both FER1L4 and p73 knockdown enhanced the migration and invasion rate of colorectal cancer cells. FER1L4 also plays a critical role in p73-mediated cell-cycle arrest and apoptosis. FER1L4 sponged the expression of miR-1273g-3p, which, in turn, increased PTEN expression, leading to cell-cycle arrest. RNA in situ hybridization revealed the down-regulation of both p73 and FER1L4 expression in a metastatic colon cancer tissue as compared with non-metastatic tissue. Collectively, we impart conclusive proof that p73 exerts its anti-metastatic properties by inducing lncRNA FER1L4 in response to genotoxic stress.

INTRODUCTION
Non-coding RNAs do not possess protein-coding properties but function as important regulatory transcripts in biological processes. These consist of transfer RNAs (tRNAs), microRNAs (miRNAs), ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), and long non-coding RNAs (lncRNAs) (Struhl, 2007). LncRNAs are usually more than 200 nucleotides in length and lack an open reading frame encoding a protein, and they differ from any known class of non-coding RNA (Wapinski and Chang, 2011). LncRNAs control gene expression at various levels that encompass transcriptional, post-transcriptional, and translational modulation (Batista and Chang, 2013;Fernandes et al., 2019;Geisler and Coller, 2013;Rinn and Chang, 2012;Xu et al., 2014). A mounting number of studies reveal that lncRNAs portray pivotal roles in tumorigenesis and may be utilized in the diagnosis of cancers Geisler and Coller, 2013;Naz et al., 2021;Xu et al., 2014). Various studies demonstrate that the deregulated lncRNAs have a major role in the pathogenesis of many human cancers, such as bladder cancer, colorectal cancer, gastric cancer, breast cancer, and esophageal cancer (; Shao et al., 2014;Tang et al., 2013). However, the attributes and roles of lncRNAs in tumorigenesis have remained chiefly obscure. It is of utmost importance to explore novel lncRNAs as biomarkers for therapeutic intervention. p73 belongs to the p53 tumor suppressor pedigree and plays an important role in tumor suppression (Dö tsch et al., 2010;Jost et al., 1997;Melino et al., 2002;Yoon et al., 2015). The significance of p73 in tumor suppression is accentuated by the observation that p73 knockout mice are extremely susceptible to unconstrained tumor development. As a result of its vigorous anti-proliferative function, p73 is maintained at low quantities in unstressed cells. Under genotoxic stress, p73 binds to its target genes and restrains cell proliferation by activating cell-cycle arrest and apoptosis followed by senescence. The p73 gene generates several isoforms by using its extrinsic (P1) or an alternative intrinsic (P2) promoter (Logotheti et al., 2013). The mechanisms of function of p73 isoforms stretch past those of a quintessential transcription factor and hold paramount significance. It is well known that TAp73 isoforms are tumor suppressive, whereas the N-terminal-lacking isoforms exert oncogenic properties (Stiewe et al., 2002). We have also recently confirmed the anti-metastatic role of p73 through the regulation of Navigator-3, a microtubule-binding protein (Uboveja et al., 2020). Taken together, p73 circuits can impose functional redundancy to p53 pathways as they administer different methods of reinstating flawed p53-regulated mechanisms in cancer cells. Nevertheless, escalating evidence indicates that these familiar p73 target genes may not be totally accountable for mediating p73 function in tumor suppression. Hence, elucidating novel p73 target genes are of paramount importance for the extensive interpretation of how p73 wields its tumor suppressive function.
Even though the literature on the p73 transcriptional circuit chiefly concentrates on protein-coding genes, it has been progressively pointed out that p73 is also able to transcriptionally modulate non-coding RNA (ncRNA) members. These involve microRNAs (miRNAs) and many p53-regulated long non-coding RNAs (lncRNAs). Diverse miRNAs have been empirically pinpointed to interact with p73 in cancer circuits. For instance, miR193A is known to be a direct p73 transcriptional target and miR193A inhibition reinstates p73-mediated cisplatin responsiveness in chemo-resistant tumors such as squamous cell carcinomas and osteosarcomas (Jacques et al., 2016;Ory et al., 2011). miR-323 is another p73-inhibiting miRNA, which can suppress p73 and accentuate prostate cancer growth and docetaxel resistance (Gao and Zheng, 2018). Analogous to miR-323, miR-1180 induces p73 to curb apoptosis in Wilms' tumors (Jiang et al., 2018). However, no study has been carried out to explore lncRNAs as possible targets of p73 transcriptional regulation. The current understanding of lncRNAs is rather restricted as compared with protein-coding genes. Nevertheless, mounting observations indicate that lncRNAs may serve as chief regulatory molecules that regulate gene expression in an array of human diseases, including cancer (Huarte, 2015;Liu et al., 2014;Yoon et al., 2013).
Fer-1-like family member 4 (FER1L4) lncRNA is a pseudogene and located on chromosome 20 in humans. It does not possess protein-coding capabilities but, instead, produces a 6,717-bp ncRNA that is majorly expressed in embryonic and adult tissues. It has been reported that lncRNA FER1L4 is down-regulated in gastric cancer (Xia et al., 2015), hepatocellular carcinoma (Wu et al., 2017), colon cancer (Yue et al., 2015), and endometrial carcinoma (Qiao and Li, 2016) and functions as a tumor suppressor in these cancers. In endometrial carcinoma, lncRNA FER1L4 suppresses cell proliferative ability and cell cycle via targeting PTEN (Qiao and Li, 2016). In glioblastoma, lncRNA FER1L4 is up-regulated and controls the glioma cells tumorigenicity (Ding et al., 2017). LncRNA FER1L4 can predict prognosis in hepatocellular carcinoma (Wu et al., 2017). However, the clinical importance and biological function of FER1L4 in colon cancer are still uncertain and need to be probed further.
In this study, we report that long non-coding RNA FER1L4 functions as a direct p73 transcriptional target and is up-regulated under genotoxic distress in a p73-dependent manner. The discovery of p73-binding sites in the FER1L4 promoter region pinpoints that FER1L4 expression is under direct regulation by p73, which was further established by chromatin immunoprecipitation (ChIP) and sitedirected mutagenesis experiments. LncRNA FER1L4 also plays a chief role in p73-mediated cell-cycle arrest and apoptosis, as revealed by cell cycle analysis and Annexin-V/PI and TUNEL apoptosis assays. FER1L4 knockdown augments cell proliferation, migration, and invasion in a p73-dependent manner. We provide evidence that FER1L4 sponges miR-1273g-3p, thus inhibiting its oncogenic role. Furthermore, RNA in situ hybridization (RNA-ISH) performed in non-metastatic and metastatic human colon cancer tissue samples confirms that FER1L4 and p73 levels are significantly down-regulated in metastatic samples as compared with non-metastatic tumor tissue samples. Collectively, our findings provide strong evidence that lncRNA FER1L4 serves as a key player in mediating tumor suppressive function of p73.
To further check whether p73 and FER1L4 levels get induced by other genotoxic drugs, HCT116p53 À/À p73 +/+ , HCT116p53 À/À p73kd, H1299p53 À/À p73 +/+ , and H1299p53 À/À p73kd cells were exposed to doxorubicin (10 mM) treatment for 24 h. p73 and FER1L4 expression was then checked by quantitative real-time PCR. The expression of both the genes was found to be significantly decreased in p73kd cells as compared with p73 +/+ cells upon doxorubicin treatment upto 24 h ( Figure 1D). Collectively, our results demonstrate that p73 expression is a prerequisite for increase in FER1L4 expression.
In our literature survey, we came across lncRNA MALAT1, which is known to play a crucial role in cancer cell progression and tumorigenesis. We wanted to check whether MALAT1 is also regulated by p73 in a similar manner as FER1L4 lncRNA. In this direction, we conducted a qPCR to check the expression levels of p73, FER1L4, and MALAT1 in HCT116p53 À/À p73 +/+ , HCT116p53 À/À p73kd, H1299p53 À/À p73 +/+ , and H1299p53 À/À p73kd cells. Our results clearly demonstrated that FER1L4 expression levels decreased significantly in p73kd cells as compared with p73wt cells ( Figure S1F). However, MALAT1 expression levels remained unchanged in both p73wt and p73kd cells, indicating that MALAT1 lncRNA is not regulated by p73. To further delineate whether p53 also plays a role in FER1L4 regulation, specific shRNA was used to abrogate p73 expression in HCT116p53 +/+ cells (control cell line) and pooled puromycin-resistant population was selected. Knockdown of p73 was confirmed by western blotting ( Figure S1G). FER1L4 mRNA levels in control and p73kd cells subjected to etoposide treatment were measured by quantitative real-time PCR. FER1L4 mRNA levels were observed to be notably decreased in p73kd cells even when p53 is present ( Figure S1H), indicating that p53 protein does not alter FER1L4 expression levels.

FER1L4 suppresses colorectal cancer cell proliferation
To ascertain the role of FER1L4 in cell proliferation, its expression was down-regulated by transfecting HCT116p53 À/À p73 +/+ cells with siRNA targeting FER1L4 (si-FER1L4). After 24 h, we evaluated the silencing efficiency under genotoxic stress (20 mM) by quantitative real-time PCR ( Figure S2A) and QuantiGene ViewRNA fluorescent RNA-ISH ( Figure S2B), and the results demonstrated that FER1L4 expression was significantly lower in the si-FER1L4 groups as compared with the control group (HCT116p53 À/À p73 +/+ cells transfected with scrambled siRNA). We further carried out a CCK-8 assay to investigate the role of FER1L4 in cell proliferation. Cell growth curves revealed that down-regulating FER1L4 expression in HCT116p53 À/À p73 +/+ cells by using specific siRNA against FER1L4 notably increased cell growth as compared with the control cells (HCT116p53 À/À p73 +/+ cells transfected with scrambled siRNA) ( Figure 2A). Also, p73kd (HCT116p53 À/À p73 +/+ cells transfected with specific shRNA against p73) cells followed a similar trend and increased cell growth as compared with the control cells (HCT116p53 À/À p73 +/+ cells transfected with scrambled shRNA) ( Figure 2A). 5-Ethynyl-2 0 -deoxyuridine (EdU) staining was carried out to further confirm the anti-proliferative ability of FER1L4, and the results demonstrated . Continued HCT116p53 À/À p73 +/+ cells transfected with scrambled shRNA), and p73kd (HCT116p53 À/À p73 +/+ cells transfected with specific shRNA against p73) were subjected to etoposide treatment (20 mM) for the indicated time points. Cells were treated with propidium iodide and quantified by flow cytometry. The data presented are representative of three independent experiments.
Pinpointing putative p73-binding sites in the lncRNA FER1L4 promoter region As our data indicate that FER1L4 is a p73-inducible gene and p73 originally acts as a transcription factor that induces its target DNA sequences, our next step was to check whether p73 interacts with the promoter of FER1L4. Hence, we scanned the human FER1L4 genomic region upstream and downstream of the Transcription Start Site (TSS) for prospective p73-binding sites by employing the TFBind software (tfbind.hgc.jp) and JASPAR database (jasper.genereg.net). One perfect p73-binding site comprises two 10-bp half-sites, 5 0 -RRRC(A/T)(A/T)GYYY, where C(A/T)(A/T)G acts as the core sequence with purine (R) and pyrimidine (Y) bases functioning as flanking sequences. The two half binding sites are separated by a spacer of upto 13 bases ( Figure S5A). Fourteen presumed p73 half binding sites (BS) were observed in the upstream region (À4,002 bp) and nine sites were observed in the downstream region (+2,900 bp) with respect to the TSS ( Figure S5B). In order to ascertain the binding of p73 with these prospective binding sites, the upstream region was further divided into three parts: U1 (À4,002 to À3,107; 896 bp), U2 (À2,539 to À1,825; 715 bp), and U3 (À1,680 to À844; 837 bp), and the downstream region was further divided into two parts: D1 (+1,092 to +1,691; 600 bp) and D2 (+2,116 to +2,900; 785 bp) ( Figure S5C).

p73 directly interacts with FER1L4 promoter
To identify the functional p73-binding site located in FER1L4 promoter, five fragments that consist of these prospective p73-binding sites-U1, U2, U3, D1, and D2-were cloned upstream of a luciferase reporter gene in pGL4.2 firefly luciferase reporter vector. These different reporter constructs were then transfected into HCT116p53 À/À cells along with pRL-TK, which acts as an internal loading control and encodes Renilla luciferase ( Figure 4A). Luciferase activity was quantified post 48 h etoposide treatment. The empty pGL4.20 luciferase vector was utilized as a negative control. Reporter constructs carrying the U1 (À4,002 to À3,107) and U2 (À2,539 to À1,825) regions demonstrated notably higher luciferase activity under genotoxic stress as compared with the control ( Figure 4B). In contrast, constructs carrying U3, D1, and D2 regions demonstrated negligible alteration in luciferase activity upon treatment with etoposide as compared . Continued (C) Cell lysates from HCT116p53 À/À p73 +/+ (Control -HCT116p53 À/À p73 +/+ cells transfected with scrambled siRNA), FER1L4kd (HCT116p53 À/À p73 +/+ cells transfected with siRNA against FER1L4), and p73kd (HCT116p53 À/À p73 +/+ cells transfected with specific shRNA against p73) cells were collected and western blotting was conducted for the indicated proteins. b-Actin was taken as loading control. Densitometry analysis is also shown. (B) Luciferase reporter assay in untreated and etoposide-treated (20 mM, 48 h) HCT116p53 À/À p73 +/+ cells transfected with empty pGL4.20 vector or pGL4.20-FER1L4 promoter-reporter constructs and pRL-TK. The results are recorded as mean G SD of three independent experiments (unpaired two-tailed Student's t test, ***p < 0.001) (C) Schematic portrayal of two prospective p73-binding sites, BS1 (À3781 to À3761) located in U1 region and BS2 (À2381 to À2362) located in U2 region of FER1L4 promoter (D and E) ChIP qRT-PCR conducted in etoposide-treated (20 mM) HCT116p53 À/À p73 +/+ (Control) and HCT116p53 À/À p73kd (p73kd) cells for the desired time points with p73 antibody and primers particularly designed for BS1 binding site located in U1 region (D) and BS2 binding site located in U2 region (E) of FER1L4 promoter. b-Actin was kept as a negative control and p21 as a positive control. The results are recorded as mean G SD of three independent experiments (paired two-tailed Student's t test, **p < 0.01, ***p < 0.001). The three samples (Input, IgG, and p73 pull down) for negative control b-actin (0 h) were run on the same gel but under another 15-well comb so that the size of the combs remained the same. They were merged in the final picture iScience Article with the control ( Figure 4B). We discovered two complete p73-binding sites BS1 (À3781 to À3761) and BS2 (À2,381 to À2,362) present in the U1 and U2 regions of FER1L4 promoter, respectively ( Figure 4C), having least mismatches with the p73 complete binding sequence.
To further attest the direct binding of p73 with FER1L4 promoter in vivo and to pinpoint the binding site, chromatin immunoprecipitation (ChIP) assays were conducted. HCT116p53 À/À p73 +/+ and p73kd cells were subjected to etoposide (20 mM) treatment for indicated time points, following which ChIP was conducted using p73 antibody. qRT-PCR was performed using the immunoprecipitated DNA and the total input DNA along with primers that flanked the putative p73-binding site BS1; BSS1 (À3,781 to À3,761) located in the U1 region of FER1L4 promoter to give rise to an expected 185-bp product and BS2 site; BSS2 (À2,381 to À2,362) located in the U2 region of FER1L4 promoter, to produce a 220-bp product. B-actin was used as a negative control, and p21 was kept as a positive control. Endogenous p73 was identified to interact specifically with BS1 ( Figures 4D and S6A) as well as with BS2 ( Figures 4E and S6B), and the fold enrichment amplified with an increase in the duration of etoposide treatment. In contrast, no noteworthy increase in binding to FER1L4 promoter (BS1 and BS2) was detected in etoposide-treated p73kd cells. These results clearly demonstrated that p73 binds the predicted sequences (BS1 and BS2) directly in the FER1L4 promoter to induce FER1L4 transcription in vivo.
p73-binding sites in FER1L4 promoter were confirmed by site-directed mutagenesis To validate the binding of p73 to BS1 and BS2 sites, luciferase reporter constructs having mutations in the core sequence of BS1 ( Figure 5A) and BS2 ( Figure 5B) p73-binding sites were created by nucleotide substitutions. Luciferase assay was conducted 24 h post etoposide treatment. Mutation of CAAG to AGAT Etoposide-treated (20 mM, 24 h) HCT116p53 À/À p73kd (p73kd) cells were transfected with FER1L4 overexpression plasmid, and quantitative real-time PCR was carried out to quantify FER1L4 mRNA levels in comparison with etoposidetreated HCT116p53 À/À p73 +/+ cells (Control) and etoposide-treated HCT116p53 À/À p73kd cells transfected with empty pCDH plasmid. The results were normalized with b-actin. The results of three independent experiments are recorded as mean G SD (unpaired two-tailed Student's t test, ***p < 0.0001) (B) Wound healing experiment (B) and its quantification (C) utilizing etoposide-treated (20 mM) HCT116p53 À/À p73 +/+ (Control -HCT116p53 À/À p73 +/+ cells transfected with scrambled siRNA), si-FER1L4 (FER1L4kd -HCT116p53 À/À p73 +/+ cells transfected with siRNA against FER1L4), HCT116p53 À/À p73 +/+ (Control -HCT116p53 À/À p73 +/+ cells transfected with scrambled shRNA), HCT116p53 À/À p73kd (p73kd -HCT116p53 À/À p73 +/+ cells transfected with specific shRNA against p73), and HCT116p53 À/À p73kd cells transfected with FER1L4 overexpression plasmid. Images were captured every 12 h to measure the width of the wound using live cell imaging system. The data presented are representative of three ll OPEN ACCESS iScience 25, 103811, February 18, 2022 iScience Article in the first half-site of BS1 binding site (U1 mut1) resulted in a 2.5-fold decrease in the luciferase levels. An equivalent mutation in the second half-site of BS1 binding site (U1 mut2) led to a 2.9-fold depletion in the luciferase levels even in the absence of etoposide. A 5.75-fold decrease in the luciferase levels of U1 mut1 was detected after etoposide treatment, whereas a 5.25-fold decrease was detected in the case of U1 mut2 after etoposide treatment, indicating that both the half-sites are crucial for p73 binding. Mutating both the half-sites (U1 mut1+2) invalidated almost entirely the binding of p73 as negligible luciferase activity was detected ( Figure 5C). Likewise, in the absence of etoposide treatment, mutating CATG to AGTT in the first half-site of BS2 binding site (U2 mut1) resulted in no alteration in the luciferase levels. Mutating CATG to AGTT in the second half-site of BS2 binding site (U2 mut2) also led to negligible change in the luciferase levels. In the etoposide-treated cells, mutations in the two half-sites of BS2 gave similar results with 7.5-fold and 7.4-fold decrease in luciferase levels with U2 mut1 and U2 mut2, respectively. Mutating both the halfsites (U2 mut1+2) abrogated practically entire p73 responsiveness ( Figure 5C), indicating that both the halfsites of BS2 are equally necessary for p73 binding. These results attest that p73 binds specifically with both BS1 and BS2 binding sites located in FER1L4 promoter to regulate its expression.

Knockdown of FER1L4 advances Epithelial to Mesenchymal Transition (EMT)
Next, we assessed whether FER1L4kd has any effect on the expression of biomarkers involved in invasion and migration, including EMT transition markers such as E-cadherin, N-cadherin, snail, fibronectin, and vimentin. In this direction, HCT116p53 À/À p73 +/+ (Control -HCT116p53 À/À p73 +/+ cells transfected with scrambled siRNA), si-FER1L4 (FER1L4kd -HCT116p53 À/À p73 +/+ cells transfected with siRNA against FER1L4), HCT116p53 À/À p73 +/+ (Control -HCT116p53 À/À p73 +/+ cells transfected with scrambled shRNA), and p73kd (HCT116p53 À/À p73 +/+ cells transfected with shRNA against p73) cells were given etoposide (20 mM) treatment for 24 h and western blotting was conducted. Results portrayed that FER1L4 knockdown cells exhibited noteworthy down-regulated protein expression levels of epithelial marker E-cadherin, whereas the expression of the mesenchymal markers, such as Snail, Vimentin, N-cadherin, and Fibronectin, was remarkably up-regulated in FER1L4kd cells as compared with the control cells. In a similar way, p73 knockdown cells also exhibited reduced E-cadherin expression and enhanced N-cadherin, Snail, Vimentin, and Fibronectin expression ( Figures 6F, and S7). Collectively, these results corroborate the observation that FER1L4 limits the migration and invasion ability of colorectal cancer cells and plays a key part in inhibition of EMT in a p73-dependent manner.

FER1L4 functions as a competing endogenous RNA and sponges miR-1273g-3p in colorectal cancer
In recent times lncRNAs have been implicated to be engaged in several molecular pathways, for instance, RNA splicing, DNA replication, and regulation of gene expression. Additionally, role of lncRNAs as competing endogenous RNA (ceRNA) has emerged as a new pathway (Fatima et al., 2015;Zhang et al., 2016). It is known that FER1L4 acts as a ceRNA in gastric cancer (Xia et al., 2015), yet lncRNAs function as a ceRNA could sponge multiple miRNAs to modulate several targets. Hence, two target prediction tools RegRNA2.0 and lncRNASNP2 were utilized to assess potential miRNAs that could have prospective binding sites for FER1L4. Bioinformatic results indicated seven miRNAs with potential binding sites for FER1L4 ( Figures S8A-S8G). To further screen which miRNA actually binds at the 3 0 UTR of FER1L4 in vitro, reporter plasmids carrying the predicted miRNA wild-type or mutant binding sites (pmirGLO-FER1L4-Wt or pmirGLO-FER1L4-Mt) were generated. These reporter plasmids were co-transfected with the individual miRNA mimics or negative control miRNA (miR-NC) mimic and dual luciferase reporter assay was carried out ( Figure S8H). Among the target miRNAs, miR-1273g-3p was chosen for our study, based on the decreased luciferase activity observed by miR-1273g-3p in the presence of pmirGLO-FER1L4-WT compared with the control (cells transfected with miR-NC), whereas pmirGLO-FER1L4-Mt was unable to produce this suppression ( Figures S8H, 7A and 7B). A complementary binding site is present in the 3 0 UTR region of FER1L4 and miR-1273g-3p ( Figure 7A). These results demonstrated that miR-1273g-3p directly binds to FER1L4-binding sites, resulting in down-regulation of the luciferase activity. Transfection of p73 +/+ cells with miR-1273g-3p under stressed conditions failed to inhibit the etoposide-induced overexpression of FER1L4 ( Figure 7C), suggesting that miR-1273g-3p is working downstream of FER1L4.
On the other hand, when FER1L4 levels decrease in p73kd and FER1L4kd cells, it is unable to sponge the miR-1273g-3p, resulting in its increased levels. To further confirm the direct interaction of FER1L4 and miR-1273g-3p, RNA immunoprecipitation (RIP) experiments were employed in HCT116p53 À/À p73 +/+ cell extracts using p73 antibody. As shown in Figure S9B, both lncRNA FER1L4 and miR-1273g-3p were specifically enriched in p73 pull down samples, but not in the control IgG, suggesting that miR-1273g-3p is a bona fide lncRNA-FER1L4-targeting miRNA.

RNA in situ hybridization investigation of FER1L4 and p73 mRNA expression levels in nonmetastatic and metastatic colon cancer tissues
To attest the positive correlation between FER1L4 and p73, 15 samples each of human non-metastatic and metastatic colorectal cancer (CRC) tissues were assessed for FER1L4 and p73 mRNA expression by RNA-ISH (Table S1). The p73 protein is located mainly in the nuclei of the cancer cells, whereas FER1L4 is expressed mainly in the cytoplasm of the cancer cells. The p73 expression level was remarkably higher in non-metastatic CRC tissue samples as compared with metastatic colon cancer tissue samples ( Figure 8A-compare a and c, b and d; Figure S10A). Similarly, FER1L4 expression was significantly enhanced in non-metastatic CRC tissue samples as compared with metastatic CRC tissue samples ( Figure 8B-compare a and b, c and d; Figure S10B). Association studies were also carried out to ascertain the correlation between p73 and FER1L4 expression in non-metastatic and metastatic tissue samples, respectively ( Figures 8C and 8D). We conducted scoring of the expression pattern on a scale of 1-4 based on the intensity of staining. 1All of 15 cases of non-metastatic CRC portrayed enhanced intensity (2 or 3) ll OPEN ACCESS iScience 25, 103811, February 18, 2022 iScience Article for both FER1L4 and p73 expression. On the other hand, 9/15 cases of metastatic CRC portrayed decreased intensity (0 or 1) for both FER1L4 and p73 expression. Based on our results of p73kd and FER1L4kd cell lines and RNA-ISH, we anticipate that this significant down-regulation of p73 and FER1L4 mRNA levels could augment metastasis in colon cancer.

DISCUSSION
Over the past few years, research on the role of microRNAs in regulating malignant diseases has governed the field of non-coding RNA regulation (Borel et al., 2012), but the consequences of lncRNA in the tumorigenesis of colon cancer are still unknown to a large extent. Accumulating evidences suggest that a iScience Article plethora of lncRNAs could be utilized as diagnostic biomarkers and therapeutic targets in human cancers and portray oncogenic or tumor suppressor functions in human cancer pathogenesis. For example, colon cancer-associated transcript-2 (CCAT-2) expression is enhanced in breast cancer and reduction of CCAT2 expression inhibits cell proliferation and invasion of both MCF7 and MDA-MB 231 breast cancer cells (Cai et al., 2015). The tumor-suppressive role of lncRNA DREH (Down-regulated expression by HBx) is regulated by alterations in the architecture of the cytoskeleton as a result of binding to vimentin and inhibition of vimentin expression. Knockdown of DREH in hepatocellular carcinoma (HCC) cells through RNAi enhances migration and invasion . The expression of Homeobox transcript antisense intergenic RNA (HOTAIR) corresponds with metastasis and bad prognosis in breast, colon, and lung cancer (Chrisholm et al., 2012;Kogo et al., 2011;Loewen et al., 2014). LncRNA LET (Low expression in tumor) expression is decreased in HCC and corresponds with metastasis (Yang et al., 2011(Yang et al., , 2013. Its mechanism of action depends on binding to NF90, a double-stranded RNA-binding protein, causing ubiquitinylation and degradation of NF90. All these findings indicate that lncRNAs function as regulators and influence various biological circuits and portray a pivotal regulatory role in carcinogenesis. Hence, identification of novel lncRNAs and understanding the prospective molecular pathways would enhance the advancement of lncRNA-directed diagnostics and therapies against cancers. LncRNA FER1L4 has been disclosed to be engaged in initiation and development of several cancers by modulating cancer cell proliferation, apoptosis, and metastasis (;; Ding et al., 2017;Fatima et al., 2015;Liu et al., 2014;Qiao and Li, 2016;Wu et al., 2017;Xia et al., 2015;Yue et al., 2015). It was initially identified as existing in gastric cancer and is down-regulated in gastric cancer tissues as compared with surrounding normal tissues (Song et al., 2013). Wu et al. (2017) demonstrated that FER1L4 exerts a tumor inhibitory action on liver cancer by suppression of miR-106a-5p expression. Qiao and Li (2016) demonstrated that FER1L4 subdues cancer cell proliferation by modulating PTEN levels in endometrial carcinoma. A recent study also proposed that FER1L4 induces osteogenic differentiation of human periodontal ligament stromal cells through miR-874-3p and vascular endothelial growth factor A (Huang et al., 2020). However, the regulation and underlying mechanism of tumor-suppressive role of FER1L4 in colorectal cancer was unexplored.
In our study, we attested for the first time the association of FER1L4 and p73 tumor suppressor gene with colon cancer progression. As a tumor suppressor, p73 modulates several crucial cellular pathways, including cell cycle progression, apoptosis, genomic stability, metabolism, and metastasis (Allocati et al., 2012;Napoli and Flores, 2017;Nicolai et al., 2015;Ozaki et al., 2010;Satija and Das, 2016;Yoon et al., 2015). Our laboratory also recently identified that p73 exerts its anti-metastatic role through up-regulation of Navigator-3, a microtubule binding protein, and inhibits the migration, invasion, and metastasis of colorectal cancer cells (Uboveja et al., 2020). But still the mechanism of p73 in CRC metastasis is unclear and needs to be dissected further. In line with this direction, our study bestows conclusive proof that p73 transcriptionally regulates long non-coding RNA FER1L4 and exerts its tumor-suppressive effects through up-regulation of FER1L4 under genotoxic stress.
Our study reveals the molecular mechanisms aiding the crucial function of p73 in restriction of metastasis by regulation of FER1L4. Identification of two prospective p73-binding sites in the FER1L4 promoter region instigated us to investigate whether p73 modulates FER1L4 expression. HCT116p53 À/À p73 +/+ cells subjected to etoposide activated endogenous p73, with an associated enhancement in the mRNA levels of FER1L4. In addition, luciferase reporter experiments demonstrated that the prospective p73-binding sites in FER1L4 promoter are functional and initiate transcription in a p73-regulated manner. Essentially, other constructs that either did not possess p73-binding sites or carried mutations within the prospective site demonstrated no p73-dependent transcriptional activation of reporter gene, indicating that the predicted p73-binding sites are actually required for p73-mediated transcriptional activation of FER1L4. The association of p73 with FER1L4 promoter under in vivo conditions was indisputably confirmed by ChIP experiments.
The functional importance of FER1L4 regulation by p73 was ascertained by conducting wound healing experiments and Transwell migration and invasion assays. These assays revealed that FER1L4 knockdown increased colon cancer cell migration and invasion in a p73-dependent way. Essentially, FER1L4 functions downstream of p73 and specifically regulates cell migration as in p73kd cells, the migration rates were similar to that of FER1L4kd cells. EMT is a phenotypic change in which epithelial cells lose their polarity and cell adhesion property and undergo various biochemical alterations, becoming motile non-polarized iScience Article mesenchymal cells with migratory and invasive properties (Boyer et al., 2000;Vu and Datta, 2017). A broadly trusted marker for EMT is the loss of E-cadherin (Vleminckx et al., 1991) and up-regulation of N-cadherin (Hazan et al., 2000), Vimentin (Hendrix et al., 1997), Snail (Batlle et al., 2000), and Fibronectin (Park and Schwarzbauer, 2014). FER1L4kd and p73kd cells demonstrated a significant decrease in the expression of E-cadherin, and noteworthy up-regulation of the mesenchymal markers as compared with that of the control cells, thereby indicating that FER1L4 plays a significant role as a metastasis deterrent in a p73-dependent manner. Collectively, these results firmly indicate that p73 exerts its anti-metastatic activity through up-regulation of lncRNA FER1L4.
LncRNAs carry out regulatory activities in cancers through a sophisticated circuit, the ceRNA regulatory circuit by which lncRNAs can sponge miRNAs and then liberate the miRNA target gene to augment either tumor progression or suppression. Furthermore, reports have ascertained that FER1L4 acts as a ceRNA to exert its regulatory function in multiple cancers. For instance, FER1L4 acts as a ceRNA to modulate PTEN expression by functioning as a sponge for miR-106a-5p in gastric cancer (Xia et al., 2015). Another study has reported that FER1L4, functioning as a ceRNA for miR-18a-5p, carries out its anti-cancer function by regulating the expression of PTEN in osteosarcoma (Fei et al., 2018). FER1L4 has also been found to suppress oncogenesis by interacting with miR-106a-5p in colon cancer (Yue et al., 2015). Furthermore, as a ceRNA, FER1L4 can sponge multiple miRNAs to carry out its regulatory activity. Hence, we screened bioinformatics databases and identified seven miRNAs that could possess FER1L4-binding sites. Based on literature review, miR-1273g-3p was chosen as a potential candidate that FER1L4 can interact with. miR-1273g-3p has an oncogenic role, which has been confirmed in many studies. For instance, it has been validated that miR-1273g-3p augments proliferation, migration, and invasion of colorectal cancer cells by inducing ERBB4/PIK3R3/mTOR/S6K2 signaling mechanism . To understand the relationship between FER1L4 and miRNA-1273g-3p we carried out series of experiments, viz., (1) a dual luciferase assay, which demonstrated that, in the presence of miR-1273g-3p, the luciferase levels dropped with constructs carrying FER1L4wt region compared with that of cells transfected with constructs carrying mutated FER1L4 (FER1L4-Mt) region in the presence of miR-1273g-3p; (2) under genotoxic stress, transfection with miR-1273g-3p failed to down-regulate the levels of FER1L4; (3) both the p73kd and FER1L4kd cells showed high levels of miR-1273g-3p under genotoxic stress, whereas in p73 +/+ cells, levels of miR-1273g-3p were low; and (4) when p73kd cells are transfected with FER1L4 overexpression plasmid, it leads to a significant decrease in miR-1273g-3p levels even when transfected with miR-1273g-3p mimics as FER1L4 is able to sponge the expression of miR-1273g-3p. Taken together, all these experiments strongly suggest that FER1L4 regulates the levels of miR-1273g-3p and not vice versa. To further support our observations, we investigated the expression of PTEN, a known downstream target of miR-1273g-3p, in p73 +/+ , p73kd, and FER1L4kd cells. We found that PTEN expression was significantly increased in etoposide-treated p73 +/+ cells, whereas low levels of PTEN were observed in p73kd and FER1L4kd cells. Furthermore, there was a significant increase in PTEN expression levels in p73kd cells transfected with FER1L4 overexpression plasmid even in the presence of miR-1273g-3p mimics. We conclude that increased levels of FER1L4 in p73 +/+ cells sponge miR-1273g-3p and thereby inhibit its oncogenic functions, whereas low levels of FER1L4 in FER1L4kd cells will result in high levels of miR-1273g-3p, leading to the increased proliferation rates and decreased apoptosis.
Our previous study assessed p73 expression in tumor tissues of patients with CRC and identified reduced p73 expression in CRC tumor tissue as compared with normal colon tissue (Kotulak et al., 2016;Uboveja et al., 2020). In this direction, we compared the mRNA expression of FER1L4 and p73 by RNA-ISH in metastatic and non-metastatic colon cancer tissues and determined whether there exists co-expression of FER1L4 and p73 as suggested in our study. Notably, we detected reduced mRNA expression of both FER1L4 and p73 in metastatic tissues as compared with non-metastatic tissues, further confirming our observations. Additionally, a significant association between FER1L4 and p73 mRNA levels was observed in both non-metastatic and metastatic tissue samples. These findings suggest that FER1L4 expression is positively governed by p73 overexpression in colorectal cancers to restrict cancer cell metastasis, whereas inhibition of p73 results in decreased FER1L4 expression in colorectal cancer cells, leading to enhanced metastatic activity of cancer cells.
Collectively, our study gives conclusive proof that p73 acts as a positive transcriptional modulator that directly regulates lncRNA FER1L4 and FER1L4 is crucial for p73-mediated suppression of colorectal cancer cell invasion, migration, and metastasis. iScience Article

Limitations of the study
Our in vitro data reveal that, upon genotoxic stress, p73 tumor suppressor gene gets upregulated and increases the levels of FER1L4 lncRNA. The positive correlation between p73 and FER1L4 lncRNA has been established by various experiments in p53 À/À colorectal cancer cell line and by RNA-ISH experiment in metastatic and non-metastatic colorectal cancer tissue samples. Later on, we further prove that FER1L4 lncRNA sponges the oncogenic potential of miRNA-1273g-3p by various experiments in p53 À/À colorectal cancer cell line. However, this negative correlation between FER1L4 lncRNA and miR-1273g-3p should have been further confirmed in colorectal cancer patient samples, which could not be completed and included in this article.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available owing to privacy or ethical restrictions.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
Plasmids generated in this study are available upon request from the lead contact, Daman Saluja (dsalujach1959@gmail.com).

Data and code availability
-All data produced in this study are included in the published article and its supplemental information, or are available from the lead contact upon request -For each analysis performed, the software and version used have been detailed in the key resources

Cell lines
HCT116p53 À/À and HCT116p53 +/+ cell lines were a kind gift from the lab of Bert Vogelstein, Johns Hopkins University, Maryland, US. The cell lines were maintained in Dulbecco Modified Eagle's Medium (DMEM) complete with fetal bovine serum (Invitrogen) and 100 U/mL Penicillin Streptomycin at 37 C in humidified air with 5% CO₂. They were frequently examined for mycoplasma contamination. HCT116p53 À/À p73kd cell line was created by transfecting pBABEU6 vector containing shRNA against p73 and puromycin-resistant cells were selected. The sequences used for shRNA are as follows: TAp73 (5 0 -GGCATGACTACATCA -3 0 ), scrambled for TAp73 (5 0 -GCCAGACTCGTTTACATGA -3 0 ). HCT116p53 À/À FER1L4kd cell line was created by transfecting four siRNA's (Qiagen, Catalogue number-1027418) against FER1L4. HCT116p53 +/+ p73kd cell line was created by transfecting pBABEU6 vector containing shRNA against p73 and puromycin-resistant cells were selected for further study. Every transfection was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer's protocol. Cells were permitted to grow to $50% confluency prior to treatment with etoposide (Sigma) for the indicated time periods.

Luciferase assays
Cells were seeded in a 12 well plate and grown overnight. The different FER1L4 promoter constructs (500 ng) were co-transfected with Renilla luciferase construct pRL-TK (500 ng) using Lipofectamine 2000 reagent (Thermo Fischer Scientific) as per manufacturer's protocol. Cells were exposed to etoposide (20 mM) treatment six hours post-transfection. After incubation for 24 hours, cells were given two washes with PBS and lysed in passive lysis buffer (300 mL/well) supplied by the Dual-Luciferase Reporter Assay System (Promega). The lysate was used to analyze firefly and renilla luciferase activities using luminometer.
Firefly luciferase values were then normalized to Renilla luciferase. The error bars indicate mean G SD of three independent experiments conducted.

Cell counting kit 8 (CCK-8) assay
Measurement of cell viability was conducted by Cell Counting Kit-8 (DojinDO, Japan) assay. Briefly, 1 3 10 3 transfected cells were seeded into a 96-well plate. CCK-8 solution (10 mL) was added into each well at different time points (24 h, 48 h and 72 h), for an additional 4 hours at 37 C. The absorbance value was calculated in a microplate reader at 450nm wavelength.

Ethynyldeoxyuridine (EdU) assay
Cell proliferation was measured by EdU assay kit (Abcam) as per manufacturer's instructions. Cells were seeded in 6-well plates on a coverslip at a density of 5 3 10 3 cells per well post-transfection. Following this, the cells were treated with 20 mM EdU labelling medium and were cultured at 37 C under 5% CO₂ for 4 hours. The cultured cells were fixed for 30 min with 4% paraformaldehyde (pH= 7.4). Cells were then washed and treated with permeabilization buffer for 20 min at RT. EdU reaction cocktail (200 mL) was then added to react with EdU for 30 min followed by addition of Hoechst 33342 (100 mL) to the cells. Fluorescent microscopy was utilized to analyze the percentage of EdU positive cells in five random fields per well.

Cell cycle analysis
The transfected cells at 60% confluency were serum deprived for 24 hours. The G0 phase synchronous cell population thus procured was subjected to etoposide (20 mM) treatment for various time points. Post the required time interval, cells were washed with cold PBS, centrifuged and fixed in 70% (v/v) ethanol at 4 C. Ethanol was then removed by washing the cells with cold PBS twice. Following this, cells were treated with PBS containing RNaseA (0.005 mg/mL) at 37 C for 30 min. Cells were then treated with propidium iodide (PI) (0.1 mg/mL) and incubated in dark for 15 min at room temperature. The cells distribution in different phases of the cell cycle was measured on FACScalibur using CellQuestPro software (Becton Dickinson, USA).

QuantiGene ViewRNA ISH cell assay
HCT116p53 À/À p73 +/+ (Control) and HCT116p53 À/À p73 +/+ FER1L4kd (FER1L4kd) cells were seeded (0.1 million) onto cover slips in 6-well plates. The cells were subjected to etoposide (20 mM) treatment for 24 hours and then fixed with 4% formaldehyde. The cells were then permeabilized with protease solution, followed by incubation with label probes against FER1L4 for 3 hours at 40 C. The cells are then treated with pre-amplifier mix, amplifier mix and label probe according to the manufacturer's protocol. Finally the cells were stained with DAPI and viewed by fluorescent microscopy.
Terminal deoxynucleotidyltransferase dUTP nick end labelling (TUNEL) assay DNA fragmentation caused by endonucleases is one of the events that occur during apoptosis resulting in the formation of multiple 3 0 hydroxyl ends. These ends are labelled with fluorescein-12-dUTP at 3 0 -OH DNA ends using Terminal Deoxynuleotidyl Transferase (TdT) and are identified directly by fluorescent microscopy. Staining was performed as per the manufacturer's instructions (Promega).

Statistics
Every statistical analysis was conducted using Prism software (Graphpad Prism5). All data are expressed as mean G (SD) of atleast three independent experiments. Two-tailed Student's t-test was utilized for calculating significance between two groups. A one way analysis of variance was utilized in case of multiple group testing. A two-tailed value of p < 0.05 was considered statistically significant and *p < 0.05,**p < 0.01, ***p < 0.001. The statistical significance of p73 and FER1L4 expression were analysed by Pearson c 2 test.


Effects of proportions of carbohydrates and fats in diets on mucin concentration and bile composition in gallbladder of dogs

The associations of diet compositions with mucin secretion in gallbladder have not been investigated in dogs. This study aimed to examine the effects of a low-carbohydrate diet (LC) and a low-fat diet (LF) on bile mucin concentration and composition of gallbladder bile in six clinically healthy beagle dogs. After feeding of both diets, the bile mucin concentration was significantly decreased. In addition, there were significant decreases in the concentrations of taurochenodeoxycholic acid in bile, which is considered to promote mucin secretion, after feeding of both diets. The present study suggested that the proportions of carbohydrate and fat in diet affect the composition of gallbladder bile in dogs.

ABSTRACT. The associations of diet compositions with mucin secretion in gallbladder have not been investigated in dogs. This study aimed to examine the effects of a low-carbohydrate diet (LC) and a low-fat diet (LF) on bile mucin concentration and composition of gallbladder bile in six clinically healthy beagle dogs. After feeding of both diets, the bile mucin concentration was significantly decreased. In addition, there were significant decreases in the concentrations of taurochenodeoxycholic acid in bile, which is considered to promote mucin secretion, after feeding of both diets. The present study suggested that the proportions of carbohydrate and fat in diet affect the composition of gallbladder bile in dogs.

KEYWORDS: bile acid, canine, low-carbohydrate diet, low-fat diet, mucin
Gallbladder diseases in dogs include gallbladder mucocele, bile sludge, and cholelithiasis. Gallbladder mucocele is a disease of excessive accumulation of viscous mucus in the gallbladder, and bile sludge is an accumulation of echogenic material without acoustic shadowing in the gallbladder. Cholelithiasis is a disease with stones in the gallbladder due to crystallization or precipitation of substances in the bile. In humans, cholesterol gallstones are common, but gallstones observed in dogs are primarily composed of mucin, bilirubin and calcium [13]. Because the main component of these mucus and substances has been found to be mucin in dogs, the excessive mucin secretion has been thought to be associated with the occurrences of gallbladder disease [2,3,17].
Mucin is secreted by the mucosal epithelium of the gallbladder and is essential for protection of the mucosal epithelium [5,17,18]. Mucin secretion is affected by inflammation of the gallbladder and changes in the composition of bile acids in humans [23]. The major bile acids in dogs include taurocholic acid (TCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA), and it has been reported that TDCA and TCDCA promote mucin secretion from the gallbladder epithelium [14,24]. Therefore, it is reasonable to hypothesize that increased TDCA and TCDCA concentrations in the gallbladder of dogs would be associated with the occurrence of canine gallbladder diseases.
A previous study in dogs showed that feeding a high-fat and high-cholesterol diet increased TCDCA concentrations in bile, although feeding a low-fat diet decreased TDCA concentrations and increased tauroursodeoxycholic acid (TUDCA) concentrations [11]. Actually, previous studies also showed the associations between diet and formation of gallstones, and feeding a high-carbohydrate diet was shown to be associated with formation of pigment gallstone in hamsters [16] and in humans [19]. Also in prairie dogs, feeding a high-carbohydrate diet induced increased TCDCA concentrations and formation of bile sludge and gallstone [1]. In dogs, low-protein, high-cholesterol, high-carbohydrate, and methionine-deficient diet induced bile sludge and pigment gallstones [8]. However, there has been no study that investigated the effects of the proportion of carbohydrates and fats in diet on composition of gallbladder bile in dogs.
Therefore, the purpose of this study was set to examine the changes in the concentration of mucin and the composition of bile acids in gallbladder bile after feeding of two diets with different proportions of carbohydrates and fats.
Six beagle dogs kept in our laboratory were used in this study. Four dogs were castrated male and two dogs were spayed female, the median age was 7 (1-7) years old, and the median body weight was 10.0 (8.2-12.0) kg. The signalment of each dog is shown in Supplementary Table 1. Before this study, they were fed a commonly available total nutritional diet twice a day for 1 month. They were kept in cages and the temperature of the room was kept at 25°C. All dogs had no clinical sign and underwent physical examination, complete blood counts, plasma biochemical analysis, thoracic X-ray examinations and abdominal ultrasonographic examination. Since bile sludge is observed in the gallbladders of clinically healthy dogs [4], dogs with bile sludge were not excluded in the present study. Five dogs were considered healthy based on the results of these examinations, and one dog continued to show elevated alkaline phosphatase (ALP). The dog with elevated ALP was not excluded from the present study, because the histopathological examination of the liver tissue of the dog did not show any abnormalities. All experimental and animal care procedures complied with the policies outlined in the Guide to Animal Use and Care of the University of Tokyo (Approval No. P20-069). This study was designed as an open, randomized, 2 × 2 crossover trial. Six dogs were arbitrarily allocated into two groups of three dogs each and fed a low-carbohydrate diet (LC) or low-fat diet (LF) for 4 weeks, and they were fed the other diet for 4 weeks after a 4-week washout period according to the previous studies [11,16]. The LC and LF used in this study were commercially available therapeutic diets (Canine glycobalance canned dog food, Gastrointestinal low fat wet; Royal Canin Japon, Tokyo, Japan). The diet that was used during washout period was also a commercially available therapeutic diet that was not low-carbohydrate or low-fat one (Select Skin Care; Royal Canin Japon) (Supplementary Table 2). Caloric intake during the feeding period was constant, and the animals were fed twice a day.
Before and after each 4-week feeding periods, we measured body weight of dogs, collected blood and gallbladder bile samples, and measured the volume of bile sludge. Five mL of blood samples were collected and plasma was immediately separated to measure ALP, alanine aminotransferase (ALT), γ-glutamyltranspeptidase (GGT), aspartate aminotransferase (AST), calcium (Ca), glucose, blood urea nitrogen (BUN), creatinine, lipase, albumin, total bilirubin, total cholesterol (T.Cho) and triglyceride using Fuji Dri-Chem 5000V (Fujifilm Corp., Tokyo, Japan).
During the collection of gallbladder bile, dogs were sedated with medetomidine (0.01 mg/kg, i.v.) and midazolam (0.1 mg/kg, i.v.), and gallbladder bile was obtained via ultrasound-guided cholecystocentesis [10]. After 12-18 hr of fasting, all bile in the gallbladder was collected. The concentration of mucin in bile was measured using Fecal Mucin Assay Kit (Cosmo Bio Ltd., Tokyo, Japan) [6]. The concentrations of each bile acid, total bile acids, bilirubin, phospholipid, cholesterol, and calcium in the gallbladder bile were measured at a commercial testing laboratory (SRL, Tokyo, Japan). Bile acids and bilirubin, phospholipids and cholesterol, and calcium were measured by high-performance liquid chromatography, spectrophotometry, and atomic absorption spectrophotometry, respectively.
For the measurements of the volume of bile sludge, long axis image of the gallbladder was obtained by ultrasonography (HI VISION Ascendus, HITACHI, Tokyo, Japan) under sedation after 12-18 hr of fasting, and the proportions of bile sludge area in the gallbladder area were calculated using software (Image J version 1.53, https://imagej.nih.gov/ij/). Then, the volumes of bile sludge were approximated by multiplying the calculated proportions of bile sludge by the volumes of the gallbladder that were calculated according to the elliptic method; V=0.53×a×b×c, V: volume of the gallbladder, a: maximum length of the gallbladder in the long axis, b: maximum width of the gallbladder in the long axis, c: maximum depth of the gallbladder in the short axis [7].
The results of blood examinations, bile mucin concentration, proportion of each bile acid, and volume of bile sludge were compared between before and after feeding periods of each food by Wilcoxon signed rank test using statistical software (JMP, SAS Institute, Cary, NC, USA). Values of P<0.05 were considered significant.
No significant change in body weight was observed after 4-week feeding period of LC or LF, although two dogs (Dog 3 and Dog 6) had mild decrease of appetite and diarrhea for several days during the feeding period of LC.
Results of blood examinations showed that ALP (P=0.03), GGT (P=0.03), and BUN (P=0.03) were significantly decreased after feeding of LC, and T.Cho was significantly decreased after feeding of LF (P=0.03) (Supplementary Table 3).
The median bile mucin concentration was significantly decreased from 37.5 (21.0-52.6) mg/dL to 12.0 (4.9-19.1) mg/dL after feeding of LC (P=0.03, Fig. 1). Also after feeding of LF, the median bile mucin concentration was significantly decreased from 21.7 (15.9-37.7) mg/dL to 17.2 (11.8-31.1) mg/dL (P=0.03, Fig. 1). When examined the changes in the concentrations of bile acids, there was no significant change in the concentration of TCA after feeding of LC or LF ( Fig. 2A). However, the concentrations of TCDCA (P=0.03, Fig. 2B), which promotes mucin secretion, and total bile acid (P=0.03, Supplementary Table 4) were significantly decreased after feeding of LC. There were also significant decreases in the concentrations of TCDCA (P=0.03), TDCA (P=0.03), which also promotes mucin secretion, TUDCA (P=0.03), taurolithocholic acid (TLCA) (P=0.03), and total bile acids (P=0.03) in bile after feeding of LF ( Fig. 2B and 2C, and Supplementary Table 4), and the concentration of glycochenodeoxycholic acid (GCDCA) were significantly increased (P=0.03) after feeding of LF (Supplementary Table  4). These results indicated the possibility that the decrease of TCDCA and TDCA in bile resulted in the decrease of mucin secretions into gallbladder bile. Previous studies showed that 1465-1468, 2022 CYP7a1 and CYP8b1 were associated with bile acid synthesis [9,22], and CYP7a1 mRNA expression was significantly increased by feeding of high-fat and high-cholesterol diet [11]. Considering these findings, it was suggested that the changes in the proportions of carbohydrates and fats in the diets might induce the changes in the expression of molecules related to the bile acid synthesis pathway, such as CYP7a1 and CYP8b1. Additionally, the expression of MUC5ac, one of the major canine mucin subtypes, was increased in bile of dogs with gallbladder mucocele compared to healthy dogs [12]. Since we did not examine the concentrations of each mucin subtype in bile in this study, it is needed to examine the associations of changes in diet compositions with the expressions of each mucin subtype. The decrease of total bile acid concentration was thought to be derived from the decreases in the concentrations of TDCA or TCDCA after feeding of the diets, because these bile acids account for 31.5-35.5% of total bile acids in dogs [24]. The increase in the concentration of GCDCA in bile was also observed after feeding of LF, which was not observed after feeding of LC. In this study, glycine was contained in LF but not in LC and maintenance diet, and the difference in the intake amounts of glycine might result in the difference of changes in the concentrations of GCDCA, which is glycine-conjugated bile acids. In the present study, the concentrations of TUDCA and TLCA were significantly decreased after feeding of LF. On the other hand, a previous study showed that the concentrations of TUDCA decreased and those of TLCA increased after the feeding of high-fat and high-cholesterol diet [11].
Although the causes of the differences in the results were unclear, the composition of fat in the diet may affect the concentrations of TUDCA and TLCA in gallbladder bile. There was a tendency to decrease in the bile calcium concentration (P=0.06) (Supplementary Table 4) after feeding of LC, and there were significant decreases in the concentrations of bilirubin (P=0.03), and calcium (P=0.03) in bile after feeding of LF (Supplementary  Table 4). There was no significant change in blood bilirubin concentration after feeding LF (Supplementary Table 3). In humans, it has been reported that ionized calcium concentration in bile is positively correlated to calcium concentration in bile [20] and the concentrations of ionized calcium in bile increases following to the increase of bile mucin concentration [20]. Therefore, it was possible that the decrease of bile mucin concentration induced the decrease of bile calcium concentration also in dogs after feeding of LC and LF. Bilirubin is present in the blood as unconjugated forms and in the gallbladder as conjugated forms after it undergoes glucuronide conjugation in the liver [1]. Therefore, the results in this study suggested that the glucuronic acid conjugation capacity in the liver may have decreased after feeding of LF due to uncovered mechanisms. However, other mechanisms should be also investigated because blood bilirubin concentration did not significantly change after feeding of LF.
We examined the changes in the proportions of each bile acid in gallbladder bile, and it was shown that the proportions of TCA (P=0.03) and glycocholic acid (GCA) (P=0.03) in bile were significantly increased and that of TCDCA was significantly decreased (P=0.03) after feeding of LC (Supplementary Fig. 1A and 1B, and Supplementary Table 5). After feeding of LF, the proportions of TCA (P=0.03) and GCDCA (P=0.03) were significantly increased, and those of TCDCA (P=0.03), TUDCA (P=0.03), and TLCA (P=0.03) were significantly decreased (Supplementary Fig. 1A and 1B, and Supplementary Table 5). However, there was no significant change in the proportion of TDCA after feeding of LC or LF (Supplementary Fig. 1C). The increase of the proportion of TCA after feeding of LC and LF and that of GCA after feeding of LF were thought to be derived from the decreased concentrations of total bile acids without significant changes of TCA and GCA concentrations.
Change in the bile sludge volumes were various among dogs, and when statistically analyzed, there was no significant change in the volume of bile sludge after feeding of LC or LF (Supplementary Fig. 2). In humans, it has been suggested that various factors other than mucin secretions such as Ca 2+ and Na + may be involved in the formation of bile sludge [1,15,20,21], and further studies are needed to investigate the detailed mechanisms that induce the formations of bile sludge in dogs.
There are several limitations in this study. The proportions of both carbohydrates and fats were different between LC and LF, and it was unclear which of the two components had greater effects on the results obtained in this study. It should be also noted that the proportion of protein was relatively high in both LC and LF. Therefore, further studies using diets where the proportions of either of carbohydrate or fat are changed are needed to clarify the effects of carbohydrates or fats in diets on the gallbladder of dogs. It is also a limitation in this study that floating mobile bile sludge in the gallbladder may not be accurately captured as bile sludge by the software. In addition, the proportions of bile sludge in the gallbladder were estimated based on the long axis image alone. Therefore, 1465-1468, 2022 further studies are needed to examine more accurate changes in the bile sludge volumes after feeding of LC and LF.
In conclusion, this study showed that both LC and LF decreased the concentration of mucin in gallbladder bile, accompanied by a decrease in the concentration of the bile acids that promote mucin secretion in gallbladder. In the future, it is needed to conduct long-term feeding trials in dogs with bile sludge or gallbladder diseases to examine the preventive or therapeutic benefits of LC or LF in canine gallbladder disease.


Leukemic Stem Cell: A Mini-Review on Clinical Perspectives

Hematopoietic stem cells (HSCs) are known for their ability to proliferate and self-renew, thus being responsible for sustaining the hematopoietic system and residing in the bone marrow (BM). Leukemic stem cells (LSCs) are recognized by their stemness features such as drug resistance, self-renewal, and undifferentiated state. LSCs are also present in BM, being found in only 0.1%, approximately. This makes their identification and even their differentiation difficult since, despite the mutations, they are cells that still have many similarities with HSCs. Although the common characteristics, LSCs are heterogeneous cells and have different phenotypic characteristics, genetic mutations, and metabolic alterations. This whole set of alterations enables the cell to initiate the process of carcinogenesis, in addition to conferring drug resistance and providing relapses. The study of LSCs has been evolving and its application can help patients, where through its count as a biomarker, it can indicate a prognostic factor and reveal treatment results. The selection of a target to LSC therapy is fundamental. Ideally, the target chosen should be highly expressed by LSCs, highly selective, absence of expression on other cells, in particular HSC, and preferentially expressed by high numbers of patients. In view of the large number of similarities between LSCs and HSCs, it is not surprising that current treatment approaches are limited. In this mini review we seek to describe the immunophenotypic characteristics and mechanisms of resistance presented by LSCs, also approaching possible alternatives for the treatment of patients.

INTRODUCTION
Hematopoietic stem cells (HSCs) are located in the bone marrow (BM) and are responsible for sustaining and regenerating the hematological system. It is estimated that in a human organism, 1x10 6 blood cells are produced every second. This feature comes from the ability of self-renewal together with a high proliferative rate and pluripotency of these cells. It is also worth mentioning the ability to resist apoptosis, necrosis and genotoxicity produced by reactive oxygen species (ROS) that HSCs have (1)(2)(3)(4)(5)(6)(7)(8)(9).
Most of the time, the HSCs are quiescent, at G0 phase of the cell cycle, depending on glucose to carry out their metabolic activities. However, these cells, when receiving the stimuli through severe situations, can quickly enter the cell cycle through activation of genetic factors. It begins with a positive control carried out in part by mTORC1 under the action of CDK6, which in the G0 phase is in low expression or accompanied by inhibitors such as p57 or p18 (8,(10)(11)(12)(13).
HSCs enters the cell cycle, and therefore, their metabolic activities start to have mitochondrial oxidative phosphorylation as a source of energy due to the increase in energy demand. This metabolic alteration consequently triggers a series of proteins, such as histone and DNA modifying enzymes, which are fundamental for the epigenetic changes carried out by the modulation of key transcription factor activity. After their activation, HSCs generate multipotent progenitors that are then committed to a cell lineage and gradually differentiate until they become mature and specialized cells (8,(10)(11)(12)(14)(15)(16).
Due to aging, HSCs lose their regenerative ability and may undergo a process called age-related clonal hematopoiesis (ARCH). In this process, mutations acquired over time continue to be transmitted to their successors, giving rise to cells with mutations. Patients with ARCH are more likely to develop leukemias, however not all cells in this process will be related to the leukemic process. It is known that the presence of certain mutations is related to the severity factor of this cell, as mutations in TP53 and U2AF1 genes are associated with pre-leukemic stem cells, and mutations in DNMT3A and TET2 genes have a lower risk regarding transformation of malignancy (12,(17)(18)(19).
In this study, we investigated clinical trials in extended literature that focused their efforts on the identification of LSCs in different types of leukemia and we discussed their clinical outcome and the perspectives of new therapies.

PRE-LEUKEMIC STEM CELL AND LEUKEMIC STEM CELL
The constant accumulation of mutations occurring in HSCs due to ARCH or other agents can stimulate the transformation of HSC into a pre-leukemic tumor cell (pre-LSCs). Although mutations are present in these cells, it is still possible for them to continue to give rise to healthy cells. However, pre-LSCs continue to accumulate mutations for years as well as significant clonal expansion until, after a long period, this cell acquires malignant characteristics, becoming a leukemic stem cell (LSC) (20)(21)(22)(23). Despite the similarities between these two types of cells, it is still possible to make differentiations, mainly genetic differentiations, where it is observed that pre-LSCs do not have mutations associated with leukemia (12,24,25).
LSCs are recognized by their stemness features such as drug resistance, self-renewal, and undifferentiated state. They were initially pointed out by Lapidot and colleagues in 1994 (34). This rare population of resistant cells is believed to be at the origin of leukemia relapses. Their quiescent state and their self-renewal capacity makes it possible to leukemia repopulating cells, despite their low frequency (35)(36)(37)(38)(39).
LSCs are present at low levels in BM, being found in only 0.1%, approximately. This makes their identification and even their differentiation difficult since, despite the mutations, they are cells that still have many similarities with HSCs (20). Although the common characteristics, LSCs are heterogeneous cells and have different phenotypic characteristics, genetic mutations, and metabolic alterations. This whole set of alterations enables the cell to initiate the process of carcinogenesis, in addition to conferring drug resistance and inducing relapses (36,37,40).
During tumor progression, cancer cells continuously acquire genetic changes, and the fittest, most proliferative cells are selected for giving rise to distinct tumor subclones, which is known as clonal hematopoiesis (CH) (41)(42)(43)(44). The clonal hematopoiesis of indeterminate potential (CHIP) refers to the presence of at least one driver mutation in hematopoietic cells of peripheral blood, without hematological malignancy. It is associated with increased risk of cancers, particularly in myeloid neoplasms, and chronic inflammatory diseases. The phenomenon of CHIP becomes very common in the population of people aged ≥80 years. That is explained by the accumulation of somatic mutations in HSCs, which occurs in an age-dependent manner (45,46).
Clones evolve through the interaction of selectively advantageous 'driver' lesions, selectively neutral 'passenger' lesions and deleterious lesions. Driver lesions or mutations are the mutations that increase fitness and confer a clonal growth advantage. The neutral mutations, also known as passenger mutations, are accumulated in these cells but do not confer any fitness advantage. Neutral evolution of these passenger mutations can also shape clonal evolution, notably by a phenomenon called genetic drift, in which the allele frequencies of a mutation change over time. In addition, when both driver and passenger mutations occur in the same cell, the passenger mutations increase their allele frequency with the driver mutations, which is a phenomenon called hitchhiking that also participate in clonal evolution (47,48).
The recognition of the important role of clonal hematopoiesis and clonal evolution in tumor initiation, disease progression and relapse have profound implications for the diagnosis and treatment of these malignancies. Additionally, the advent of new technologies may facilitate the definition of the molecular determinants and underlying mechanisms of clonal evolution in leukemia, which could provide targeted, individualized therapies for leukemia patients (49,50).

CHARACTERIZATION OF LEUKEMIC STEM CELLS
A major challenge in studying LSCs is identifying a possible unique cell surface antigen phenotype, from which it would be possible to develop a targeted and much more specific treatment than the treatments currently used. Furthermore, due to the heterogeneity of the different types of leukemias, there is a lot of variation in the antigens found on the surface of these cells, becoming even more difficult to identify a specific marker that is not expressed in normal cells, or has a different expression pattern, density, or distribution (51)(52)(53)(54).
Some common stem cell's markers are CD34, CD117 and HLA-DR, which expressions predict lower rate of complete remission (CR). In addition, the principal surface antigens of the myeloid lineage are CD13, CD33, CD14, CD15 and CD11b. Mostly, the expression of these markers have not yet showed any prognostic significance or are associated with a poorer outcome, such as reduction of CR, period of remission and survival. Although, cells that expresses CD15 usually presents a higher CR rate. Another marker found in myeloblasts is CD56, which expression is also reported as a poorer prognostic factor (55) As an example, we can point out that normal HSC constitutively expresses CD34 + and CD38antigens, in addition to others such as Thy-1 + , c-kit + and IL-3Ra. Much of the LSC population immunophenotypically resemble certain normal hematopoietic progenitor populations by also expressing CD34 + and CD38besides others surface markers (56)(57)(58) Despite these difficulties, a great number of cell surface markers have been identified that are upregulated on FIGURE 1 | Pathways of malignancy in hematopoiesis and its characteristics. Aging and exposure to hazardous environmental agents lead to accumulation of DNA damage and mutations in hematological precursor cells, inducing a pre-leukemic stem cell (pre-LSC) phenotype. Pre-LSCs acquire proliferation advantages over normal hematopoietic stem cells (HSCs) due to mutations in genes such as DNA methyltransferase 3 alpha (DNMT3a), but still retain their capacity to promote normal hematopoiesis. However, further malignant characteristics acquired over the years may tip these cells into a proper leukemic stage. The transformation of pre-LSCs may happen through cell-specific processes, such as epigenetic modulation or new acquired mutations, or through interactions between these cells and their microenvironment, through changes in the normal growth and survival signaling pathways or due to interactions with dysfunctional stromal or mesenchymal cells that are also present in the bone marrow. After malignancy onset, leukemic stem cells (LSC) may present a variety of karyotype rearrangements, such as BCR-ABL or FLT3-ITD, that determine their malignant characteristics and tend to present immunophenotyping profiles that still resemble normal HSCs, such as CD34 + 38 -, while also overexpressing a cohort of cell-surface antigens that are highly variable between patients and even among different cell populations in the same patient.
Some studies addressing chronic myeloid leukemia (CML) LSCs demonstrated that differentially expressed antigens include CD25, CD26, IL-1RAP, which is associated with the activation of NF-kb and AKT signaling pathways, increasing proliferation of CML LSCs. In addition, the overexpression of the antigen CD25 is reported to reduce proliferation capacity of CML LSCs. Some data suggest CD25 and IL-1RAP expression are unique to LSCs of this type of leukemia (54,63,64).
The presence of LSCs is related to the rates of complete remission (CR) and general survival (OS) of patients, besides that, depending on the remaining amount, they may predispose to relapse of patients with leukemias (65). The identification of certain surface markers and molecular changes in these cells may influence the prognosis of patients, but the results of studies are still somewhat controversial. Bradstock et al. (66) pointed out that patients who expressed CD9, CD14 and CD2 in their CSLs had lower CR rates. The CD9, CD10 and CD11b markers were associated with lower OS rates, and CD11b was also related with a shorter duration of CR.
The relationship between the molecular alterations observed in patients and their prognosis is also somewhat controversial among studies. Nomdedeu et al. (68) demonstrated that the FLT3-ITD mutation had a significant influence on OS, where patients affected by this alteration had lower rates compared to those who did not have the mutation (17.9 vs 41 months). On the other, Beńéet al. (67) did not find a difference in survival between patients with and without molecular changes. However, other articles report the relationship of FLT3-ITD, MLL-PTD, RUNX1-RUNX1T1 and CBFB-MYH11 mutations with a poor outcome (71,72).
In addition, studies demonstrated a correlation between white blood cells (WBC) count and poorer prognosis. Patients with a higher WBC count were less likely to achieve CR and presented a shorter survival rate (67,73). Besides, a higher platelet count was associated with a longer survival time (67). These general findings corroborate with data found in other articles (74)(75)(76)(77).

PRESENCE OF LSCS AND RELAPSE
Even with the course of the disease, it is possible to observe that pre-LSCs and LSCs continue to evolve throughout the process, in addition to the fact that treatment often fails to reach these cells.
LSCs play a key role from development to disease relapse. Thus, its analysis and quantification can be of great importance as a prognostic factor for patients. Such processes can also assist in choosing a more targeted treatment. The identification of these cells can be performed through immunophenotyping, where it is possible to differentiate HSCs from LSCs. From this differentiation, counting methods are performed (78)(79)(80).
Recent studies point to two classifications of relapse related LSCs: the first classification is known as LSCs of committed relapse origin, where these cells are most like the diagnostic cell type and were able to evolve similarly to the diagnostic dominant clone. The second classification is the LSCs of primitive relapse origin, which are rare cells at the diagnosis of the disease and do not usually form blasts. However, these cells may show greater resistance to treatment and later clonal evolutions, causing the patient to relapse, which usually continues with the increase in the amount of LSCs and with greater heterogeneity (81,82).
Studies reveals that LSCs levels correspond to the clinical and laboratory characteristics of the patient. Due to their insufficient morphological and biochemical characterization, LSCs cannot be reliably measured in patient samples. However, as a consequence of the finite capacity of the joined stem cell niche, HSCs can act as a biomarker for LSCs numbers and help identify patients with an adverse clinical outcome (74,83,84). This is noticeable through the LSCs and the HSCs count, considering that the BM may have a limited number of cells. Therefore, LSCs and HSCs compete for niches, their values being inversely proportional, that is, the more LSCs the less HSCs. Therefore, patients who had a low LSCs load also had lower blast, platelet, and leukocyte counts. In addition to clinical features, the cells count can represent how it might respond molecularly. This is important when evaluating the chosen treatment. Therefore, lower levels of LSCs are associated with a better molecular response to treatment (85,86).
Still on the quantification of LSCs, its functionality also applies to the assessment of measurable residual disease (MRD) and is considered an effective biomarker for predicting relapses. So, in addition to being used in the diagnosis to choose the treatment, the immunophenotyping test can be used in the post-treatment phase to evaluate its effectiveness and predict the patient's survival. High values of LSCs would be associated with worse survival and low efficacy. This analysis is then performed using molecular methodologies such as quantitative real-time polymerase chain reaction (RT-qPCR) or next-generation sequencing (NGS), and flow cytometry. It is important to remember that all these methodologies for prognostic analysis and MRD are still being carried out in studies and better clarification and standardization are needed for clinical application (83,84,87,88). Table 1 is comprised of clinical trials from the past 10 years that aimed to identify biomarkers specific to LSCs that could serve as targets for targeted therapies or could be used as prognostic factors (89)(90)(91)(92)(93)(94)(95)(96)(97)(98). Most of the reported studies aim at AML treatment since LSC presence and complexity is an established risk factor for disease severity (51,56).
Studies utilizing standard chemotherapy protocols and induction therapies confirm that identified LSC markers, mainly transmembrane antigens such as CD34 and CD123, correlate with a worse patient prognosis. Increased mutation burden, lower response rates, inability to achieve CR, worst response to chemotherapy and higher incidence of relapse are some of the reported factors associated with increased presence of LSCs (89,(92)(93)(94)96).
Although resistant to most treatment strategies, the use of novel agents targeting specifically LSCs molecular pathways combined with standard treatment protocols showed some promising results for AML patients (90,91,95). Huselton et al. (90) combined dociparstat sodium (DSTAT), a drug capable of inhibiting CXCR4/CXCL12 cell adhesion molecules, with hypomethylating agent (HMA) azacitidine in an attempt to disrupt bone marrow niches where LSCs remain in a quiescent state and was able to achieve CR in patients who were previously unresponsive to treatments with HMA alone.
Riether et al. (91) identified WNT pathway as being hyperactivated in AML LSCs due to increased expression and interaction of CD70/CD27 molecules. In vitro, the use of monoclonal antibodies targeting CD70 in combination with HMAs was demonstrated to have an additive effect in inhibiting LSC growth since the use of HMAs seem to increase LSCs dependency on CD70/CD27 pathway and concurrent CD70 inhibition was able to further reduce LSCs burden when compared to monotherapies of either agent. In previously untreated AML patients, protocols combining cusatuzumab plus azacitidine induced responses in all treated patients and transcriptome analysis after treatment revealed increased expression of genes involved in pathways of inflammation, differentiation and apoptosis (91).
Lastly, Wang et al. (95) utilized compound zhebei granule (CZBG), a herbal concoction with oncologic uses in traditional Chinese medicine, that acts through mechanism such as apoptosis induction and inhibition of resistance-related drug efflux proteins, combined with standard chemotherapy to treat AML patients and a significant decrease in CD34 + CD123 + cells was observed in bone marrow niches. CZBG was also demonstrated to increase response rates in AML patients in combination with chemotherapy when compared to chemotherapy alone and to be able to reduce LSCs markers in tumor cell xenografts when combined with doxorubicin treatment (95,99,100).
In CML patients, in vitro studies indicate BCR-ABL tyrosine kinase inhibitors (TKIs) to have no efficacy over LSCs and leukemic progenitor cells (LPCs) and, while imatinib initial response rates are overwhelmingly positive, disease recurrence is usually the standard for patients after therapy discontinuation due to remaining Philadelphia-positive (Ph + ) CD34 + cells (101,102). The use of next-generation TKIs, however, seem to be more effective in reducing stem and progenitor cells in CML than imatinib and may point towards a choice for more intensive treatment options in accordance with increased LSCs and LPCs burden (103).
Pungolino et al. (97) utilized nilotinib, a second-generation BCR-ABL inhibitor, to treat newly diagnosed CML patients and observed a rapid decrease in CD34 + lin -Ph + cells in the bone marrow, with total clearance of the analyzed samples at 12

TREATMENT PERSPECTIVES
Most conventional treatments for leukemias seek to eradicate blasts, reaching cells that are in their active cycle. However, LSCs are usually in a quiescent state or protected through their molecular resistance mechanisms causing this cell to resist therapy, leading to patient relapse (104)(105)(106)(107)(108)(109)(110)(111). One of the major known factors that confer drug resistance is the overexpression of drug efflux pumps, such as the ATP-binding cassette (ABC) transporter family proteins by CSCs. Moreover, drug efflux increase is often combined with the upregulation of enzymes involved in the metabolism of anticancer agents. Therefore, enzymes and efflux transporters expressed by LSCs appear to be crucial not only for their proliferation, but also for their resistance to clinical treatments (108,(112)(113)(114)(115).
The selection of a target to LSCs therapy is fundamental. Ideally, the target chosen should be highly expressed by LSCs, highly selective, absence of expression on other cells, in particular HSCs, and preferentially expressed by a high number of patients. In view of the large number of similarities between LSCs and HSCs, it is not surprising that current treatment approaches are limited (20,81,116).
Currently, different treatment methodologies have been tested and addressed, such as the use of binding antibodies associated with different toxins to form a specific delivery vehicle for LSCs. Examples of such therapies are the use of Gemtuzumab Ozogamicin for the treatment of AML, a compound that uses an antigen against CD33, associated with a cytotoxic agent; and the inhibition of the SIRP1-a interaction with CD47 that activates innate immunity increasing the death of LSCs. In addition, CD244, CD123, LLC1 or TIM3 targets are also studied and demonstrate antileukemic efficacy in AML patients. However, one of the main difficulties in the treatment of LSCs is due to the low proliferation rate, which makes it difficult to identify the cell to start the therapy (62,81,(117)(118)(119).
Several new strategies are under development to eliminating LSCs, which may result in a better patient response. Many of the studies are associated with the use of TKIs, which to improve their effectiveness can be combined with other agents. TNF-a inhibitors combined with TKIs, for example, have shown positive results in the elimination of LSCs. Blocking of IL-1 signaling may also be a combination with TKIs, as well as blocking of signal pathways such as Wnt/b-catenin, Hedgehog, MAPK/MNK1/2, mTOR, PTEN, PP2A, Alox5 and JAK/STAT. The action of HIF-1 inhibitors associated with TKIs has been shown to reduce the survival and growth of cells in CML in murine models. The HIF-1 deletion has also been tested in in vivo and in vitro models and has been shown to inhibit CML proliferation, both without serious effects on HSCs (120)(121)(122).
Activations and gene dissections can also be used, as in the case of p53 activation and EZH2 deletion. Both technologies demonstrate promising results that enhance the eradication of LSCs when combined with TKIs (123,124). The combination of TKIs with cytarabine was also performed, demonstrating good results in AML patients. Despite the large number of tests involving TKIs, medications and methodologies have also been developing as the case of Bortezomibe. Its function is based on decreasing the expression of CDK6, an important agent in the proliferation of LSCs (120,125,126).
Undoubtedly, one of the greatest difficulties in eliminating LSCs is their resistance mechanisms. As a result, studies have specialized in finding drugs and technologies that help to overcome the resistance present in LSCs. In this scenario, research diverges in different areas such as transport proteins and signaling pathways, taking as an example Notch, Hedgehog, and Wnt/b-catenin that are describes also as responsible for drug resistance (104,(127)(128)(129). It is also worth mentioning studies focused on the epithelial-mesenchymal transition (EMT), histone acetylation, hypoxia and the BM niche (104,130,131).
The tumor microenvironment (TME) creates a niche for itself that influences not only the proliferation and differentiation of LSCs but also the response to drugs. A key factor that modulates the microenvironment and drug resistance is hypoxia, which signaling contributes to chemoresistance of CSCs by increasing the expression of ABC transporters and Aldehyde dehydrogenases, a family of intracellular enzymes, which can be used as molecular markers to identify normal stem cells (NSCs) and CSCs (104,128,(132)(133)(134). An example was demonstrated by Giuntoli et al. (134), when CML cells were grown in low oxygen concentrations and became resistant to Imatinib.
The EMT process is already known in solid tumors and has recently been explored in hematological neoplasms as well as its treatment possibilities (104,127,134,135). Thus, one of the ways is to look for drugs that can act on genes such as TRPS1, ETS2 and LSP, known to belong to this process in AML (136,137). Competition for the BM niche between LSCs and HSCs has also become a therapeutic target, transforming the environment in a more favorable way for HSCs or increasing their hematopoietic reserve. E-selectin inhibition is an example, being able to promote the displacement of HSCs and LSCs. Regarding hypoxia, the use of hypoxia-activated prodrugs such as TH-302 has already been shown to reduce the population of LSCs in an AML model (138,139).
In addition to those already mentioned, the combination of Venetoclax with Azacitidine demonstrates potential for the treatment of LSCs in AML, as it suppresses OXPHOS. Regarding gene deletion, it was observed that FOXO1 deletion through genetic or pharmacological pathways is able to inhibit the proliferation of malignant cells, which is present in LSCs and pre-LSCs, becoming a potential target. Studies aimed at the use of microRNAs mimics were also carried out and have potential, such as miR-15a/16-1 acting as a tumor suppressor acting negatively on the WT1 gene (28,81,140,141).

CONCLUSION
Foremost is important to better define the molecular and cellular biologic features of normal HSCs and LSCs, for improve the identification of possible therapeutic targets to eradicate the LSCs, that are responsible for treatment resistance and clinical relapse for most patients. It is necessary to carry out studies that correlate the quantification and immunophenotypic characterization of LSCs with clinical data and prognosis presented by patients, regarding the significance of this information pointed in the studies here presented, and due to the lack of this type of study in the literature.

AUTHOR CONTRIBUTIONS
IB, FP and CM wrote the manuscript. All authors contributed to the manuscript and were involved in revisions and proof-reading.
All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by Brazilian funding agencies: Coordination for the Improvement of Higher Education Personnel (CAPES; to CM), National Council of Technological and Scientific Development (CNPq grant number 404213/2021-9 to CM-N; and Productivity in Research PQ Scholarships to MM-F, MM, RB and AK), Cearense Foundation of Scientific and Technological Support (FUNCAP grant number P20-0171-00078.01.00/20 to FP, MM and CM-N) and we also thank PROPESP/UFPA for publication payment. the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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