<p>List of 83 miRNAs which were differentially expressed in CRC vs normal colon.</p>

* List gives the number of target gene transcripts that are hit by one or more of the miRNAs in order of decreasing number of miRNAs hitting them. **List gives the number of target gene transcripts that are hit by those miRNAs that are over-expressed in the crypt bottom in addition to hsa-mir-25 and hsa-mir-007

'Fig S1. Validation of miRNA chip results by quantitative PCR analysis. Fig S2. Analysis of differentially expressed miRNAs using target predictions. Fig S3. The relative abundance of the common predicted targets in relation to miRNA levels in CRC cell lines. Fig S4. The effect of miRNAs on mRNA expression levels of their predicted targets. Fig S5. Relative expression of four candidate microRNAs in HT29 ALDH+ cells as compared to ALDH- cells. Fig S6. miRNA23b targets 3' UTR of LGR5 and LRIG1 mRNA. Fig S7: miRNA23b affects cell proliferation. Fig S8: CaCo2 data on miRNA23b precursor and antimer effect on sphere forming ability as well as 5FU response.'

Lists of predicted gene targets of miRNAs in the upper crypt. List of predicted gene targets in the crypt bottom. GO term analysis of the top 200 predicted gene targets for both the lists.

<p>'Legends for Supplementary Figures'</p>

Abstract Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. ©2017 AACR.

HOX genes are thought to play a role in regulating stem cells (SCs) during embryological development. Aberrant expression of HOX genes is seen in many cancers, including colorectal cancer (CRC). However, the mechanisms leading to aberrant HOX gene expression in CRC are not known. Because SCs are key to colon tumorigenesis, we hypothesized that (i) expression of specific HOX genes is different in malignant colonic SCs than in normal SCs and (ii) aberrant expression of these HOX genes is necessary for the tumor initiating ability of colon cancer SCs. Using real time PCR-based array analysis, we found that expression of many HOX genes are upregulated in colon carcinomas relative to normal colonic epithelium. We previously reported a ‘unique HOX gene signature’ for the SC enriched population; two-color microarray analysis showed that two HOX genes, HOXA4 and HOXD10 are expressed in the bottom part of the colonic crypt where SCs reside. Here, we confirmed this finding, and then, using immunohistochemistry, we validated it. HOXA4 and HOX D10 were expressed at the bottom part of the normal colonic crypt. Increased expression of these genes was also seen in colon carcinoma tissues. The results for colon carcinoma tissues were validated using real time PCR. As an independent method to validate the differential expression of HOX genes, we used flow cytometry and ALDEFLUOR to isolate, from a colon cancer cell line, SCs and non-SCs. HOXA4 and HOXD10 expression was then studied using real time PCR. HOXA4 was upregulated in SCs vs non-SCs. To determine whether these genes are differentially expressed in carcinomas relative to normal tissue, we co-stained tissues with antibodies against HOXA4 or HOXD10, and with SC markers, ALDH1 or CD166. We found that SCs express these HOX genes in the bottom part of the colonic crypt compared to non-SCs. In colon carcinoma tissues, not only was there co-staining, but also, the co-staining was increased relative to normal colonic epithelium. Our results implicate expression of HOXA4 and HOXD10 genes not only in the functioning of normal SCs, but also in the contribution of colon SCs to colon tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-102. doi:1538-7445.AM2012-LB-102

Abstract Our goal is to determine how transcriptional regulatory mechanisms that control crypt cell maturation in normal colonic epithelium, when dysregulated, lead to maturation arrest, tumor stem cell (SC) overpopulation and colorectal cancer (CRC) development. Accordingly, we studied changes in maturation during colon cancer development caused by APC mutations. Quantitative mapping of crypt cell subpopulations was done on colon tissues from FAP patients using markers for stem cells (SC), proliferating cells, cell-cycle arrest, and apoptosis. In normal human colon, mapping defined the sequential transitions in colonic crypts that enterocyte subpopulations undergo during their maturation from stem-to-proliferating-to-differentiated-to-apoptotic cells, illustrating the point that maturation is a process involving both proliferation and differentiation. In normal-appearing (heterozygous-mutant) and adenomatous (homozygous-mutant) FAP crypts, SC and proliferating cell subpopulations increasingly expanded towards the crypt top, while differentiated/apoptotic subpopulations in the upper crypt contracted, indicating that crypt cell maturation becomes progressively delayed. These data indicate that there are coordinated mechanisms that regulate maturation of crypt cells in normal crypts which become dysregulated in premalignant crypts. To investigate possible transcriptional regulation of a network of genes underlying maturation, we used bioinformatic tools to identify (i) genes that are co-expressed in the proliferating cell population and (ii) transcriptional regulatory elements (TREs) statistically enriched in the promoter regions among those genes. Computational analysis revealed (i) a specific cassette of sixty-six genes that are co-expressed with the markers used for mapping the proliferating cell population in the lower colonic crypt and (ii) eleven TREs that are significantly enriched in the promoter regions among genes in this gene set. Quantitative PCR and immunohistochemistry was then used to validate that the transcription factors (TFs), which correspond to the common TREs, are differentially expressed along the normal crypt axis and in normal colonic epithelium versus malignant colon tissues. Our results indicate that dysregulation of a transcriptional regulatory network contributes to delayed crypt cell maturation, SC overpopulation, and initiation and promotion of CRC. Accordingly, restoring maturation by targeting transcriptional pathways that control expression of multiple genes involved in crypt cell maturation may be a therapeutically effective approach to CRC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4924. doi:1538-7445.AM2012-4924

Abstract Dysregulation of crypt cell proliferation and differentiation has been implicated in the development of colorectal cancer (CRC). It is believed that human colonic crypt renewal involves programmed cellular proliferation initiated by a few (6-8) stem cells residing at or near the bottom of the crypt, and that stem cell (SC) overpopulation may be the key to CRC initiation. As part of our investigation of regulatory factors involved in the stem cell origin of CRC, we investigated the role that microRNAs (miRNAs) might play in colon carcinogenesis. miRNAs are RNAs 20-24 nucleotides long that were recently shown to modulate many cellular signaling pathways through post-transcriptional regulation of messenger RNA (mRNA) levels and thus protein synthesis. Our previous microarray analysis (368 gene chip) showed increased expression of 37 miRNAs in CRCs compared to normal colonic epithelium. Here we further evaluated miRNA expression in CRC versus purified colonic epithelium by quantitative PCR (QPCR). Total RNA was immediately isolated from tissue by the TrizolTM method. RNA was transcribed into first-strand complementary DNA (cDNA) through reverse transcription (RT). cDNA was then amplified through QPCR. Statistical analysis and plots of expression data for miRNAs were done to find miRNA genes that are differentially expressed. We found, using QPCR, five miRNAs to be differentially expressed in CRCs versus normal colonic epithelium including mir-25 and mir-198. Profiling of purified colonic crypt subregions showed that a distinct signature involving 16 differentially expressed miRNAs characterizes the crypt bottom. This miRNA signature of the crypt bottom was found to be predictive of CRC. Out of the sixteen, three miRNAs (mir-206, mir- 7 and mir-25) were independently capable to distinguish colorectal tissue from normal colonic epithelia (p<0.01). Because this subset of miRNAs is differentially expressed in the crypt bottom, where SC resides, they may be involved in regulating SC population dynamics and contribute to the SC origin of CRC. Further identification and characterization of miRNAs expressed specifically in SC and changes in their expression in CRCs will provide important information to help understand mechanisms of colon tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4247.

Background: Pancreatic ductal adenocarcinoma (PDA) is a devastating disease that killed nearly 38,000 people in the United States this past year.Objective: Treatment of PDA typically includes surgery and/or chemotherapy with gemcitabine. No reliable biomarker exists for prognosis or response to chemotherapy. Two previously proposed prognostic markers, cyclooxy-genase-2 (COX-2) and vascular endothelial growth factor (VEGF), are regulated by Hu protein antigen R (HuR), an mRNA binding protein that we have previously demonstrated to be a promising predictive marker of gemcitabine response. This study was designed to evaluate the clinical utility of HuR, COX-2, and VEGF as potential prognostic and predictive biomarkers for PDA.Methods: A tissue microarray of 53 PDA specimens from patients who underwent potentially curative pancreatic resection was analyzed. HuR, COX-2, and VEGF status were correlated with clinicopathologic and survival data. We also performed ribonucleoprotein immunoprecipitation assays using an HuR antibody to assess VEGF and COX-2 mRNA binding to HuR in pancreatic cancer cells.Results: Roughly 50% (27/53) of patients had high cytoplasmic HuR expression. These patients had worse pathologic features as assessed by T staging (P = 0.005). Only cytoplasmic HuR status correlated with tumor T staging, whereas VEGF (P = 1.0) and COX-2 (P = 0.39) expression did not correlate with T staging. Additionally, HuR status was an unprecedented positive predictive marker for overall survival in patients treated with gemcitabine, pushing median survival over 45 months in the high cytoplasmic HuR expressing patient population compared with less than 23 months in the low cytoplasmic HuR expressing patient group (P = 0.033 for log-rank test and P = 0.04 in a Cox regression model) for the low versus high cytoplasmic HuR expressing group. We also validated that mRNA transcripts for both VEGF and the gemcitabine metabolizing enzyme, deoxycytidine kinase, are specifically bound by HuR in pancreatic cancer cells.Conclusions: HuR is a useful prognostic biomarker for PDA patients as indicated by its association with higher tumor T stage. Additionally, HuR status is a robust predictor of outcome for patients with resected PDA in the setting of adjuvant gemcitabine therapy. Finally, HuR binds to VEGF mRNA implying that HuR, in part, regulates VEGF expression in PDA. This study supports the notion that HuR status should be used by clinicians for the individualized treatment of PDA in the future.

Abstract PARP-inhibitors (PARPi) are a novel class of agents that target cancer cells deficient in DNA repair. Early phase trials show promising success with PARP-inhibitors, yet drug resistance mechanisms and off-target pathways affected by drug exposure have not been explored. HuR (ELAV1) is a protein that binds and stabilizes mRNA transcripts upon certain stress stimuli. These HuR mRNA targets result in an increase in protein expression; and thus, protein function. Recently, we demonstrated that HuR binds and regulates deoxycytidine kinase, the gemcitabine metabolizing enzyme. Thus, HuR expression enhances gemcitabine efficacy against pancreatic cancer cells. We subsequently screened other chemotherapeutics that may stimulate the HuR stress response. In isogenic pancreatic cancer cell lines, stable HuR overexpression rendered cells up to 2-fold more sensitive to PARPi compared to control cells. Accordingly, siRNA knock down of HuR expression in both pancreatic and ovarian cancer cell lines caused PARPi resistance compared to a scramble-sequence control siRNA. In a pancreatic cancer cell line, MiaPaCa2, a 2.5-fold siRNA knock down of HuR mRNA expression detected by qPCR rendered cells approximately 4-fold more resistant to PARPi. Similarly, a modest siRNA knock down of HuR in an ovarian cancer cell line, A2780, rendered cells more resistant to PARPi. Immunofluorescence studies indicate that a 5 hour treatment with 75 µM PARPi directly caused HuR to transport from the nucleus to the cytoplasm, presumably moving ARE-rich mRNA cargoes after drug exposure. Focused gene expression analysis on HuR bound, immunoprecipitated RNA defined functional HuR downstream targets (including deoxycytidine kinase). Current studies will reveal and validate the importance of these downstream mRNA targets in PARP-inhibitor metabolism and efficacy. We hypothesize that HuR may be central to PARPi effectiveness and may be useful in understanding PARPi de novo drug resistance mechanisms. Future work will reveal whether HuR status can be utilized as a predictive marker for PARP-inhibitor-based therapy and if all clinically available PARP-inhibitors engage HuR's regulation of unique mRNA cargoes in cancer cells upon treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3643.