Dysregulation of beta-catenin levels and localization and constitutive activation of beta-catenin/TCF (T cell factor)-regulated gene expression occur in many cancers, including the majority of colorectal carcinomas and a subset of ovarian endometrioid adenocarcinomas. Based on the results of microarray-based gene expression profiling we found the insulin receptor substrate 1 (IRS1) gene as one of the most highly up-regulated genes upon ectopic expression of a mutant, constitutively active form of beta-catenin in the rat kidney epithelial cell line RK3E. We demonstrate expression of IRS1 can be directly activated by beta-catenin, likely in part via beta-catenin/TCF binding to TCF consensus binding elements located in the first intron and downstream of the IRS1 transcriptional start site. Consistent with the proposal that beta-catenin is an important regulator of IRS1 expression in vivo, we observed that IRS1 is highly expressed in many cancers with constitutive stabilization of beta-catenin, such as colorectal carcinomas and ovarian endometrioid adenocarcinomas. Using a short hairpin RNA approach to abrogate IRS1 expression and function, we found that IRS1 function is required for efficient de novo neoplastic transformation by beta-catenin in RK3E cells. Our findings add to the growing body of data implicating IRS1 as a critical signaling component in cancer development and progression.

The "incessant ovulation" hypothesis links increased risk for tubo-ovarian high-grade serous carcinoma (HGSC) due to more ovulations and reduced risk conferred by pre-menopausal exposures like oral contraceptive use, multiparity, and breastfeeding. However, most women diagnosed with HGSC are postmenopausal, implying age is a major risk factor for HGSC. Our mouse model for HGSC, based on tamoxifen (TAM)-induced somatic inactivation of the Brca1, Trp53, Rb1, and Nf1 (BPRN) tumor suppressor genes in oviductal epithelium, recapitulates key genetic, histopathologic, and biological features of human HGSCs. We aimed to credential the model for future efforts to define biological and risk modification factors in HGSC pathogenesis.

ARID1A, encoding a subunit of the SWI/SNF chromatin-remodelling complex, is the most frequently mutated epigenetic regulator across all human cancers. ARID1A and TP53 mutations are typically mutually exclusive. Therapeutic approaches that correlate with this genetic characteristic remain to be explored. Here, we show that HDAC6 activity is essential in ARID1A-mutated ovarian cancers. Inhibition of HDAC6 activity using a clinically applicable small-molecule inhibitor significantly improved the survival of mice bearing ARID1A-mutated tumours. This correlated with the suppression of growth and dissemination of ARID1A-mutated, but not wild-type, tumours. The dependence on HDAC6 activity in ARID1A-mutated cells correlated with a direct transcriptional repression of HDAC6 by ARID1A. HDAC6 inhibition selectively promoted apoptosis of ARID1A-mutated cells. HDAC6 directly deacetylates Lys120 of p53, a pro-apoptotic post-translational modification. Thus, ARID1A mutation inactivates the apoptosis-promoting function of p53 by upregulating HDAC6. Together, these results indicate that pharmacological inhibition of HDAC6 is a therapeutic strategy for ARID1A-mutated cancers.

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.

Somatic APC (adenomatous polyposis coli), TP53, KRAS mutations are present in roughly 80%, 60%, and 40%, respectively, of human colorectal cancers (CRCs). Most TP53 mutant alleles in CRCs encode missense mutant proteins with loss-of-function (LOF) of p53's transcriptional activity and dominant negative (DN) effects on wild-type p53 function. Missense mutant p53 proteins have been reported to exert gain-of-function (GOF) effects in cancer. We compared the phenotypic effects of the common human cancer-associated TP53 R273H missense mutation to p53 null status in a genetically engineered mouse CRC model. Inactivation of one allele of Apc together with activation of a Kras mutant allele in mouse colon epithelium instigated development of serrated and hyperplastic epithelium and adenomas (AK mice). Addition of a Trp53R270H or Trp53null mutant allele to the model (AKP mice) led to markedly shortened survival and increased tumor burden relative to that of AK mice, including adenocarcinomas in AKP mice. Comparable life span and tumor burden were seen in AKP mice carrying Trp53R270H or Trp53null alleles, along with similar frequencies of spontaneous metastasis to lymph nodes, lung, and liver. The fraction of adenocarcinomas with submucosa or deeper invasion was higher in AKP270/fl mice than in AKPfl/fl mice, but the incidence of adenocarcinomas per mouse did not differ significantly between AKPfl/fl and AKP270/fl mice. In line with their comparable biological behaviors, mouse primary tumors and tumor-derived organoids with the Trp53R270H or Trp53null alleles had highly similar gene expression profiles. Human CRCs with TP53 R273 missense mutant or null alleles also had essentially homogeneous gene expression patterns. Our findings indicate the R270H/R273H p53 mutant protein does not manifest definite GOF biological effects in mouse and human CRCs, suggesting possible GOF effects of mutant p53 in cancer phenotypes are likely allele-specific and/or context-dependent.

Clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are ovarian carcinoma histotypes, which are both thought to arise from ectopic endometrial (or endometrial-like) cells through an endometriosis intermediate. How the same cell type of origin gives rise to two morphologically and biologically different histotypes has been perplexing, particularly given that recurrent genetic mutations are common to both and present in nonmalignant precursors. We used RNA transcription analysis to show that the expression profiles of CCOC and ENOC resemble those of normal endometrium at secretory and proliferative phases of the menstrual cycle, respectively. DNA methylation at the promoter of the estrogen receptor (ER) gene (ESR1) was enriched in CCOC, which could potentially lock the cells in the secretory state. Compared with normal secretory-type endometrium, CCOC was further defined by increased expression of cysteine and glutathione synthesis pathway genes and downregulation of the iron antiporter, suggesting iron addiction and highlighting ferroptosis as a potential therapeutic target. Overall, these findings suggest that while CCOC and ENOC arise from the same cell type, these histotypes likely originate from different cell states. This "cell state of origin" model may help to explain the presence of histologic and molecular cancer subtypes arising in other organs.

If left untreated, some cervical high-grade squamous intraepithelial lesions will progress to invasive squamous cell carcinoma (SCC), but the molecular events conferring invasive potential remain poorly defined. In prior work, we identified 48 genes that were down-regulated in SCCs compared with high-grade squamous intraepithelial lesions and normal squamous epithelia. In this study, a functional screening strategy was used to identify which of these genes regulate cervical cancer cell invasion. Two independent squamous epithelial cell lines were transduced with a library of short hairpin RNAs targeting the differentially expressed genes and tested for invasion of the chick chorioallantoic membrane. PCR was used to recover specific short hairpin RNAs from cells that invaded the chorioallantoic membrane. Constructs targeting estrogen receptor 1 (ESR1) were highly enriched in the invasive cells. The short hairpin RNA-mediated inhibition of ESR1 in SCC- and precancer-derived cell lines increased invasiveness in both in vivo and in vitro assays. Conversely, restoration of ESR1 expression in ESR1-negative cervical cancer cells reduced cell invasiveness. Loss of ESR1 expression was found to accompany cervical cancer progression in an analysis of primary normal cervix, low grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and SCC specimens. Molecular mechanisms underlying down-regulation of ESR1 in invasive cervical carcinomas appear to be complex and likely heterogeneous. Our findings indicate that loss of ESR1 has a major role in mediating cervical cancer invasion and progression.

Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the β-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding β-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected β-catenin levels and some β-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected β-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis.

We generated transgenic mice in which human CDX2 gene elements control expression of a tamoxifen-regulated Cre protein (CDX2P-CreER(T2)) to allow for inducible gene targeting in intestinal epithelium. After tamoxifen dosing of CDX2P-CreER(T2) mice, Cre activity was detected in the distal ileal, cecal, colonic, and rectal epithelium, with selected crypt base, transit amplifying, and surface cells all capable of activating Cre function. Four weeks after tamoxifen dosing of CDX2P-CreER(T2) mice carrying a Cre-activated fluorescent reporter, single crypts were uniformly fluorescence positive or negative, reflecting Cre activation in crypt stem cells. Biallelic inactivation of the Apc tumor suppressor gene via the CDX2P-CreER(T2) transgene in colon epithelium led to acute alterations in cell proliferation, apoptosis, and morphology, along with mitotic spindle misorientation, β-catenin nuclear localization, and induction of the intestinal stem cell markers Lgr5 and Musashi-1 and the Sox9 transcription factor. Normal mouse colon epithelium lacks Paneth cells, a key small intestine niche cell type, and Paneth cell differentiation is dependent on Sox9 function. In Apc-deficient colon epithelium, ectopic Paneth-like cells were seen outside the crypt base, such as new crypt budding sites. Our data indicate Apc inactivation via CDX2P-CreER(T2) targeting in mouse colon epithelium is sufficient to induce adenomatous changes and the generation of Paneth-like cells from neoplastic progenitors, with potentially significant roles in colon adenoma development and progression.

One histologic subtype of ovarian carcinoma, ovarian endometrioid adenocarcinoma (OEA), frequently harbors mutations that constitutively activate Wnt/beta-catenin-dependent signaling. We now show that defects in the PI3K/Pten and Wnt/beta-catenin signaling pathways often occur together in a subset of human OEAs, suggesting their cooperation during OEA pathogenesis. Deregulation of these two pathways in the murine ovarian surface epithelium by conditional inactivation of the Pten and Apc tumor suppressor genes results in the formation of adenocarcinomas morphologically similar to human OEAs with 100% penetrance, short latency, and rapid progression to metastatic disease in upwards of 75% of mice. The biological behavior and gene expression patterns of the murine cancers resemble those of human OEAs with defects in the Wnt/beta-catenin and PI3K/Pten pathways.

While 20-30% of colorectal cancers (CRCs) may arise from precursors with serrated glands, only 8-10% of CRCs manifest serrated morphology at diagnosis. Markers for distinguishing CRCs arising from 'serrated' versus 'conventional adenoma' precursors are lacking. We studied 36 human serrated CRCs and found CDX2 loss or BRAF mutations in ~60% of cases and often together (p=0.04). CDX2Null/BRAFV600E expression in adult mouse intestinal epithelium led to serrated morphology tumors (including carcinomas) and BRAFV600E potently interacted with CDX2 silencing to alter gene expression. Like human serrated lesions, CDX2Null/BRAFV600E-mutant epithelium expressed gastric markers. Organoids from CDX2Null/BRAFV600E-mutant colon epithelium showed serrated features, and partially recapitulated the gene expression pattern in mouse colon tissues. We present a novel mouse tumor model based on signature defects seen in many human serrated CRCs - CDX2 loss and BRAFV600E. The mouse intestinal tumors show significant phenotypic similarities to human serrated CRCs and inform about serrated CRC pathogenesis.