Hmga2 mediates Lin28b effects on stem cell colony formation and enteroid proliferation.

<p>Colony formation assay of small intestine enteroids from wild-type (WT) mice (A) and <i>Vil-Lin28b</i>^<i>Med</i> mice (B). Two wells of a 24-well plate are pictured. C) Quantification of colony forming assay of WT mice and <i>Vil-Lin28b</i>^<i>Med</i> mice. D) Schematic of lentiviral vector-mediated transduction of enteroids with a “tet-on” doxycycline (dox)-inducible vector. Inducible expression of a turbo RFP reporter in the unmodified pTRIPz vector is readily observed in enteroids (H) vs. untreated cells (G). Phase contrast images of untreated and doxycycline treated enteroids are pictured in (E) and (F), respectively. Immunostaining for Hmga2 in sectioned enteroids from pTRIPz-transduced (I), pTRIPz-Hmga2-transduced (J), and pTRIPz-Hmga2-transduced plus doxycycline (K). L) Comparison of colony forming potential of cells from dissociated enteroids transduced with pTRIPz (empty), Arid3a, Hif3a, or Hmga2 vectors. Phase contrast images of colony formation assays from enteroids transduced with pTRIPz (empty) (M) or Hmga2 (N) vectors. O) Colony forming assays of non-transgenic mice (NTG) or <i>Vil-Lin28b</i>^<i>Med</i> mice with and without inactivation of one conditional Hmga2 allele using <i>Vil-Cre</i>. EdU incorporation of enteroids, as assayed by flow cytometry, transduced with pTRIPz-Hmga2 (P) or pTRIPz-Hif3a (Q) vectors. Cultures were treated with 100 ng/ml doxycycline in all experiments except in (F) and (H), which were treated with 500 ng/ml doxycycline. All experiments were performed at least in triplicate. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to pTRIPz vector controls, unless noted otherwise.</p

Survival analysis in stage II/III patients treated with 5-FU-based chemotherapy.

<p>Kaplan-Meyer curves for <b>A</b>, Disease-free survival (DFS) and <b>B</b>, overall survival (OS) in stage II/III patients according to miR-148a expression. Kaplan-Meyer curves for <b>C</b>, DFS in stage II and <b>D</b>, stage III DFS depending upon miR-148a expression.</p

Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.

<p>A) Schematic of the intestine-specific deletion of the <i>Mirlet7c-2/Mirlet7b</i> floxed locus via <i>Villin-Cre</i> and expression of Lin28b with a <i>Villin-Lin28b-ires-tdTomato</i> transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse. F) Representative section of adenocarcinoma from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.</p

Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.

<p>A) Heat map of TCGA mRNA-seq colon and rectal adenocarcinoma dataset from UCSC Cancer Genome Browser (genome-cancer.ucsc.edu) comparing expression of Let-7 target mRNAs in normal tissue (N.T.) vs. cancer. Significant up-regulation (red) or down-regulation (green) is indicated below heatmap in plots of the-log(p-value) of Benjamini-Hochberg-corrected T-tests on the y-axis. T-test results are shown for expression in tumors vs. N.T. and in tumors associated with at least one lymph node metastases vs. tumors with no associated lymph node metastases. Inverse relationships for Let-7 and target mRNAs could be discerned by plotting miRNA-seq data against mRNA-seq data for Let-7c vs. <i>HMGA2</i> (B), Let-7a vs. <i>PLAGL2</i> (C), Let-7a vs. <i>HMGA2</i> (D), and Let-7a vs. <i>IGF2BP2</i> (E). F) Taqman QPCR for mature Let-7a and Let-7b miRNAs in a cohort of colon adenocarcinomas (N = 20) indicates that Let-7a and Let-7b are down-regulated. G) Intestinal epithelial stem cell markers <i>EPHB2</i>, <i>ASCL2</i>, and <i>LGR5</i> are significantly up-regulated in colon cancer vs. normal adjacent tissues. H) Mature Let-7a and Let-7b levels are tightly correlated in these tissue specimens. I) Let-7a and Let-7b levels are inversely proportional to mRNA levels of stem cell markers <i>EPHB2</i> and <i>LGR5</i>, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} tumors, relative to WT jejunum, with a trend for up-regulation in <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} jejunum, and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} tumors by linear regression yielded Pearson correlation coefficients, with <i>Arid3a</i>, <i>Hmga1</i>, and <i>Hmga2</i> correlating very highly with expression of stem cell markers. L) HMGA2 and LGR5 expression from the TCGA mRNA-seq colon and rectal adenocarcinoma dataset exhibit significant positive correlation. Expression analysis (F-K) was performed by QPCR, normalized to <i>Hprt</i> and <i>Actb</i>, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to <i>PPIA</i> and <i>B2M</i>, with error bars representing +/–the S.E.M. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001.</p

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

<div><h3>Aim</h3><p>Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.</p> <h3>Methods</h3><p>We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.</p> <h3>Results</h3><p>Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).</p> <h3>Discussion</h3><p>MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.</p> </div

MiR-148a expression and methylation in colonic mucosa from healthy individuals and in CRC tissues from patients.

<p><b>A</b>. miR-148a expression in colonic mucosa from healthy controls (NC), and in stage II, III and IV CRCs; the number of patients (N) and median expression (Median) are listed below the graph. <b>B</b>. <i>In situ</i> hybridization for miR-148a in CRC tumors and normal mucosa, in which the chromogen stains red, and the counterstain blue. Representative photomicrographs are shown from a normal colonic mucosa (top panels), a tumor with low miR-148a expression (middle panels), and a tumor with high miR-148a expression (lower panels) at indicated magnifications. A photomicrograph is shown from a tumor with high miR-148a expression using a scramble probe as a negative control (bottom, left panel). <b>C</b>. miR-148a methylation levels in stage IV tumors. The putative promoter region of miR-148a, and the position of pyrosequencing primers are illustrated in the top panel. The scatter plot of miR-148a expression and methylation levels are shown in the bottom panel. <b>D</b>. miR-148a expression levels are shown for methylated and non-methylated CRCs in the top panel. miR-148a methylation levels are shown for tumors with high and low miR-148a expression in the bottom panel. One outlier value (the methylation level; 48%) is excluded from the methylated group in the bottom graph.</p

Univariate and multivariate analysis of miR-148a expression, methylation and overall survival in stage IV MSS CRC patients.

a<p>p<0.05.</p

Hmga1 and Hmga2 proteins are increased in invasive areas of adenocarcinomas.

<p>Immunohistochemical staining for Hmga2 (A-C, G, H) and Hmga2 (D-F, I, J), in sections from WT small intestine (S.I.) (A, D), <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} S.I. (B, E), <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} adenoma (C, F), and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} adenocarcinoma (AdenoCA) (G-J). An enlargement of a region containing invasive HMGA1-positive tumor cells from G (dotted yellow box) is pictured in H, while a region containing invasive HMGA2-positive tumor cells from I is likewise displayed in J. Pictures in A-F, H, and J are at same magnification (200x), with scale bar = 100 μm, while pictures in G and I are both at 40x, with scale bar = 250 μm.</p

Associations between miR-148a status and therapeutic response or survival in stage IV CRC patients treated with 5-FU and oxaliplatin.

<p><b>A</b>. Therapeutic response according to miR-148a expression. Complete response, CR; partial response, PR; stable disease, SD; progressive disease; PD. <b>B</b>. Kaplan-Meyer curves for progression-free survival (PFS, left panel) and OS (right panel) in stage IV patients according to miR-148a expression. <b>C</b>. Kaplan-Meyer curves for PFS (left panel) and OS (right panel) in stage IV patients according to miR-148a methylation.</p

MiR-148 expression status and clinicopathologic characteristics of CRC patients.

a<p>The difference was analyzed by Mann-Whitney U test.</p>b<p>The difference was analyzed by Fisher's exact test.</p>c<p>Proximal colon, located above splenic flexure; distal colon, located in splenic flexure or below.</p>d<p>The difference was analyzed by the chi-square test.</p