Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/− 2 vs 56.5+/− 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.
Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of β-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses1,2. Curiously, however, ‘inflammatory signature’ genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer3,4. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates5, referred to as ‘tumour elicited inflammation’6. Although infiltrating CD4+ TH1 cells and CD8+ cytotoxic T cells constitute a positive prognostic sign in colorectal cancer7,8, myeloid cells and T-helper interleukin (IL)-17-producing (TH17) cells promote tumorigenesis5,6, and a ‘TH17 expression signature’ in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival9. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier10. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.
BACKGROUND & AIMS
Adenomatous polyps are precursors to colorectal cancer (CRC), whereas hyperplastic polyps (HPPs) have a small risk of progression to CRC. Mutations in KRAS are found in ~40% of CRCs and large adenomas and a subset of HPPs. We investigated the reasons that HPPs with KRAS mutations lack malignant potential; we compared the effects of Kras/KRAS activation to those of Adenomatous polyposis coli (Apc)/APC inactivation, which promotes adenoma formation.
We activated a KrasG12D mutant allele or inactivated Apc alleles in mouse colon epithelium and analyzed phenotypes and expression of selected genes and proteins. The mouse data were validated using samples of human HPPs and adenomas. Signaling pathways and factors that contribute to Kras/KRAS-induced phenotypes were studied in intestinal epithelial cells.
Activation of Kras led to hyperplasia and serrated crypt architecture akin to that observed in human HPPs. We also observed loss of Paneth cells and increases in goblet cell numbers. Abnormalities in Kras-mediated differentiation and proliferation required mitogen-activated protein kinase (MAPK) signaling and were linked to activation of the Hes1 transcription factor. Human HPPs also had activation of HES1. In contrast to Apc/APC inactivation, Kras/KRAS activation did not increase expression of crypt stem cell markers in colon epithelium or colony formation in vitro. Kras/KRAS activation was not associated with substantial induction of p16INK4a protein expression in mouse colon epithelium or human HPPs.
Although Kras/KRAS mutation promotes serrated and hyperplastic morphological features in colon epithelium, it is not able to initiate adenoma development, perhaps in part because activated Kras/KRAS signaling does not increase the number of presumptive stem cells in affected crypts.
Colon cancer; oncogene; tumor suppressor; transgenic mice
Genetically engineered mouse (GEM) models of ovarian cancer that closely recapitulate their human tumor counterparts may be invaluable tools for preclinical testing of novel therapeutics. We studied murine ovarian endometrioid adenocarcinomas (OEAs) arising from conditional dysregulation of canonical WNT and PI3K/AKT/mTOR pathway signaling to investigate their response to conventional chemotherapeutic drugs and mTOR or AKT inhibitors.
OEAs were induced by injection of adenovirus expressing Cre recombinase (AdCre) into the ovarian bursae of Apcflox/flox;Ptenflox/flox mice. Tumor-bearing mice or murine OEA-derived cell lines were treated with cisplatin and paclitaxel, mTOR inhibitor rapamycin, or AKT inhibitors API-2 or perifosine. Treatment effects were monitored in vivo by tumor volume and bioluminescence imaging, in vitro by WST-1 proliferation assays, and in OEA tissues and cells by immunoblotting and immunostaining for levels and phosphorylation status of PI3K/AKT/mTOR signaling pathway components.
Murine OEAs developed within 3 weeks of AdCre injection and were not preceded by endometriosis. OEAs responded to cisplatin + paclitaxel, rapamycin, and AKT inhibitors in vivo. In vitro studies showed that response to mTOR and AKT inhibitors, but not conventional cytotoxic drugs, was dependent on the status of PI3K/AKT/mTOR signaling. AKT inhibition in APC−/PTEN− tumor cells resulted in compensatory up-regulation of ERK signaling.
The studies demonstrate the utility of this GEM model of ovarian cancer for pre-clinical testing of novel PI3K/AKT/mTOR signaling inhibitors and provide evidence for compensatory signaling, suggesting that multiple rather than single agent targeted therapy will be more efficacious for treating ovarian cancers with activated PI3K/AKT/mTOR signaling.
ovarian carcinoma; endometriosis; PI3K/AKT/mTOR signaling; mouse model; bioluminescence imaging
We demonstrate that rapamycin can induce regression of adenomatous polyposis coli (Apc) mutation-dependent colonic adenomas in genetically engineered mice (CPC;Apc). An endoscope was used to visualize adenomas in CPC;Apc mice weekly for 10 weeks. The lesion surface areas were measured using a distance gauge and digitally generated grid. Coronal scans were performed on magnetic resonance imaging (MRI) to localize adenomas, and tumor volumes were measured from regions of interest drawn on consecutive axial scans. Rapamycin (5 mg/kg) was administered intraperitoneally daily for 5 weeks. Endoscopy and MRI were performed weekly to monitor adenoma regression. Caliper measurements and immunohistochemistry (IHC) were performed on adenomas postmortem. Dimensions from n = 30 adenomas in n = 7 animals were measured. Adenoma surface areas on endoscopy correlated with volumes on MRI and with postmortem caliper measurements, R2 = 0.84 and R2 = 0.81, respectively. The mean adenoma doubling times on endoscopy and MRI were 0.95 ± 0.14 and 1.21 ± 0.16 weeks, respectively. The minimum detectable adenoma surface area and volume on endoscopy and MRI was 0.69 mm2 and 1.76 mm3, respectively. On histology, the rapamycin-treated adenomas showed limited regions of dysplasia. Rapamycin therapy resulted in much lower mammalian target of rapamycin signaling and cell proliferation. Lower expression of phospho-S6 and reduced numbers of Ki67-positive cells were seen on IHC compared to vehicle-treated lesions. Endoscopy can be validated by MRI as a robust methodology for quantitative monitoring of therapy, representing a promising approach for future preclinical efforts to assess utility of novel colorectal cancer prevention strategies.
High-frequency microsatellite instable (MSI-H) tumors account for roughly 15% of colorectal cancers (CRC). Therapeutic decisions for CRC are empirically based and currently do not emphasize molecular subclassification despite of the increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI CRCs using combined gene expression data sets.
Three expression/query signatures (discovery data set) characterizing MSI-H CRC were matched with information derived from changes induced in cell lines by 164 compounds, using the systems biology tool “Connectivity Map”. A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle and apoptosis effects of validated compounds were evaluated in a panel of cell lines.
Fourteen of the 164 compounds were validated as targeting MSI-H cells lines using the bioinformatics approach; Rapamycin, LY-294002, 17-AAG and Trichostatin-A were the most robust candidate compounds. In vitro results showed that MSI-H cell lines due to hypermethylation of MLH1 are preferentially targeted by Rapamycin (18.3 vs 4.4 μM, P=0.0824) and LY-294002 (15.02 vs 10.37 μM, P=0.0385) when compared to MSS cells. Preferential activity was also observed in MSH2 and MSH6-mutant cells.
Our study demonstrates that the PI3K-AKT-mTOR pathway is of special relevance in mismatch repair-deficient CRC. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.
Microsatellite instability; colorectal cancer; gene expression patterns; rapamycin; mTOR pathway; PI3K inhibitors
Chronic inflammatory disorders are often associated with an increased cancer risk. A particularly striking example of the chronic inflammation–cancer link is seen in inflammatory bowel disease, in which chronic colitis or persistent inflammation in the colon is associated with elevated risk of colorectal cancer. Animal models exploring the mechanisms by which inflammation increases the risk of colon cancer have shown that inflammatory cells, through the effects of the cytokines they produce, have a major role in promoting neoplastic transformation. In this issue of the JCI, Popivanova and colleagues demonstrate that TNF-α, through its effects on the immune system, plays a critical role in promoting neoplastic transformation in this setting (see the related article beginning on page 560). Importantly, the study also provides evidence that anti–TNF-α therapies, which are currently in clinical use, may interrupt the process.
Pregnane X Receptor (PXR), a master regulator of drug metabolism and inflammation, is abundantly expressed in the gastrointestinal tract. Baicalein and its O-glucuronide baicalin are potent anti-inflammatory and anti-cancer herbal flavonoids that undergo a complex cycle of interconversion in the liver and gut. We sought to investigate the role these flavonoids play in inhibiting gut inflammation by an axis involving PXR and other potential factors. The consequences of PXR regulation and activation by the herbal flavonoids, baicalein and baicalin were evaluated in vitro in human colon carcinoma cells and in vivo using wild-type, Pxr-null, and humanized (hPXR) PXR mice. Baicalein, but not its glucuronidated metabolite baicalin, activates PXR in a Cdx2-dependent manner in vitro, in human colon carcinoma LS174T cells, and in the murine colon in vivo. While both flavonoids abrogate dextran sodium sulfate (DSS)-mediated colon inflammation in vivo, oral delivery of a potent bacterial β-glucuronidase inhibitor eliminates baicalin’s effect on gastrointestinal inflammation by preventing the microbial conversion of baicalin to baicalien. Finally, reduction of gastrointestinal inflammation requires the binding of Cdx2 to a specific proximal site on the PXR promoter. Pharmacological targeting of intestinal PXR using natural metabolically labile ligands could serve as effective and potent therapeutics for gut inflammation that avert systemic drug interactions.
Expression of the essential EMT inducer Snail1 is inhibited by miR-34 through a p53-dependent regulatory pathway.
Snail1 is a zinc finger transcriptional repressor whose pathological expression has been linked to cancer cell epithelial–mesenchymal transition (EMT) programs and the induction of tissue-invasive activity, but pro-oncogenic events capable of regulating Snail1 activity remain largely uncharacterized. Herein, we demonstrate that p53 loss-of-function or mutation promotes cancer cell EMT by de-repressing Snail1 protein expression and activity. In the absence of wild-type p53 function, Snail1-dependent EMT is activated in colon, breast, and lung carcinoma cells as a consequence of a decrease in miRNA-34 levels, which suppress Snail1 activity by binding to highly conserved 3′ untranslated regions in Snail1 itself as well as those of key Snail1 regulatory molecules, including β-catenin, LEF1, and Axin2. Although p53 activity can impact cell cycle regulation, apoptosis, and DNA repair pathways, the EMT and invasion programs initiated by p53 loss of function or mutation are completely dependent on Snail1 expression. These results identify a new link between p53, miR-34, and Snail1 in the regulation of cancer cell EMT programs.
Ovarian endometrioid adenocarcinomas (OEAs) frequently exhibit constitutive activation of canonical WNT signaling, usually as a result of oncogenic mutations that stabilize and dysregulate the β-catenin protein. In prior work, we used microarray-based methods to compare gene expression in OEAs with and without dysregulated β-catenin as a strategy for identifying novel β-catenin/TCF target genes with important roles in ovarian cancer pathogenesis. Among the genes highlighted by the microarray studies was MSX2, which encodes a homeobox transcription factor. We found MSX2 expression was markedly increased in primary human and murine OEAs with dysregulated β-catenin compared to OEAs with intact β-catenin regulation. WNT pathway activation by WNT3a ligand or GSK3β inhibitor treatment potently induced MSX2, and ectopic expression of a dominant negative form of TCF4 inhibited MSX2 expression in ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated that β-catenin/TCF directly regulates MSX2 expression via binding to TCF binding elements in multiple regions of the MSX2 gene. Notably, ectopic MSX2 expression was found to promote neoplastic transformation of the rodent RK3E model epithelial cell line and to enhance the invasiveness of immortalized human ovarian epithelial cells in vitro and ovarian carcinoma cells in vivo. Inhibition of endogenous MSX2 expression in ovarian endometrioid cancer cells carrying a β-catenin mutation using shRNA approaches inhibited neoplastic properties of the cells in vitro and in vivo. Expression of MSX2 in selected ovarian carcinoma cells induced changes suggestive of epithelial-mesenchymal transition (EMT), but based on analysis of ovarian cell lines and primary tumor tissues, effects of MSX2 on EMT appear to be complex and context-dependent. Our findings indicate MSX2 is a direct downstream transcriptional target of β-catenin/TCF and has a key contributing role in the cancer phenotype of OEAs carrying WNT/β-catenin pathway defects.
MSX2; ovarian cancer; endometrioid; WNT signaling; β-catenin
The caudal-related homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. CDX2 heterozygous mutant mice develop colonic polyps, and loss of CDX2 expression is seen in a subset of colon carcinoma in man. Ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is frequently detected in intestinal metaplasia in the stomach and esophagus. We sought to define CDX2-regulated genes to enhance knowledge of CDX2 function. HT-29 colorectal cancer cells have minimal endogenous CDX2 expression, and HT-29 cells with ectopic CDX2 expression were generated. Microarray-based gene expression studies revealed that the Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene was activated by CDX2. Evidence that the MDR1 gene was a direct transcriptional target of CDX2 was obtained, including analyses with MDR1 reporter gene constructs and chromatin-immunoprecipitation assays. RNA interference-mediated inhibition of CDX2 decreased endogenous MDR1 expression. In various colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Over-expression of CDX2 in HT-29 cells revealed increased resistance to MDR1's known substrate, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression via binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers.
Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the
world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as
adenomatous polyps, and current endoscopic screening approaches with white light
do not detect all dysplastic lesions. Thus, new strategies to identify such
lesions, including non-polypoid lesions, are needed. We aim to identify and
validate novel peptides that specifically target dysplastic colonic epithelium
in vivo. We used phage display to identify a novel peptide
that binds to dysplastic colonic mucosa in vivo in a
genetically engineered mouse model of colo-rectal tumorigenesis, based on
somatic Apc (adenomatous polyposis coli) gene
inactivation. Binding was confirmed using confocal microscopy on biopsied
adenomas and excised adenomas incubated with peptide ex vivo.
Studies of mice where a mutant Kras allele was somatically
activated in the colon to generate hyperplastic epithelium were also performed
for comparison. Several rounds of in vivo T7 library biopanning
isolated a peptide, QPIHPNNM.
The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic
analysis. Quantitative assessment revealed the fluorescent-labeled peptide
(target/background: 2.17±0.61) binds ∼2-fold greater to the colonic
adenomas when compared to the control peptide (target/background:
1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic
(hyperplastic) epithelium of the Kras mice. This work is first
to image fluorescence-labeled peptide binding in vivo that is
specific towards colonic dysplasia on wide-area surveillance. This finding
highlights an innovative strategy for targeted detection to localize
pre-malignant lesions that can be generalized to the epithelium of hollow
In head and neck squamous cell carcinoma (HNSCC) cells, Rap1 shuttles between the nucleus and cytoplasm. Prior findings suggested that Rap1 may modulate the β-catenin-independent Wnt pathway in some settings, but the role of Rap1 in β-catenin-dependent Wnt signaling remains undefined.
Experimental Design and Results
We observed that β-catenin bound to active Rap1 in vitro and Rap1 activated β-catenin-TCF (T cell factor)-dependent transcription. Immunofluorescence studies showed that ectopic expression of Rap1 increased nuclear translocation of β-catenin. Overexpression of active Rap1 facilitated an increase in β-catenin-mediated transcription that was abrogated by dominant negative TCF4. Conversely, siRNA-mediated inhibition of endogenous Rap1 expression inhibited β-catenin/TCF-mediated transcription as well as invasion of HNSCC. Furthermore, inhibition of Rap1 expression downregulated the expresesion of MMP7, a transcriptional target of β-catenin/TCF. In HNSCC cells stably transfected with β-catenin or treated with lithium chloride or Wnt3A to stabilize endogenous β-catenin, inhibition of Rap1 expression led to decreases in the free pool of β-catenin. Immunohistochemical studies of tissue from HNSCC patients revealed that increased β-catenin intensity correlated with higher tumor stage. Furthermore, the prognostic effect of active Rap1 on tumor N-stage was found to depend on cytosolic β-catenin expression (p<0.013). When β-catenin is high, higher rap1GTP intensity is associated with more advanced N stage.
The findings suggest that Rap1 enhances β-catenin stability and nuclear localization. In addition to indicating that Rap1 has a significant role in regulating β-catenin and β-catenin-dependent progression to more advanced N-stage lesions, these data highlight Rap1 as a potential therapeutic target in HNSCC.
nucleus; Wnt signaling; TCF transcription; small GTP-binding protein
The gene encoding COMM domain–containing 1 (COMMD1) is a prototypical member of the COMMD gene family that has been shown to inhibit both NF-κB– and HIF-mediated gene expression. NF-κB and HIF are transcription factors that have been shown to play a role in promoting tumor growth, survival, and invasion. In this study, we demonstrate that COMMD1 expression is frequently suppressed in human cancer and that decreased COMMD1 expression correlates with a more invasive tumor phenotype. We found that direct repression of COMMD1 in human cell lines led to increased tumor invasion in a chick xenograft model, while increased COMMD1 expression in mouse melanoma cells led to decreased lung metastasis in a mouse model. Decreased COMMD1 expression also correlated with increased expression of genes known to promote cancer cell invasiveness, including direct targets of HIF. Mechanistically, our studies show that COMMD1 inhibits HIF-mediated gene expression by binding directly to the amino terminus of HIF-1α, preventing its dimerization with HIF-1β and subsequent DNA binding and transcriptional activation. Altogether, our findings demonstrate a role for COMMD1 in tumor invasion and provide a detailed mechanism of how this factor regulates the HIF pathway in cancer cells.
Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3β (GSK3β) by phosphorylation at serine 9. In turn, release of cytosolic membrane β-catenin with subsequent nuclear translocation promotes survival. Both GSK3β and β-catenin have been implicated in cancer cell proliferation and resistance to death.
We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3β/β-catenin axis.
Lithium chloride, RNAi-medicated silencing of GSK3β, or the expression of a kinase dead mutant GSK3β resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3β resulted in radiosensitization of Panc1 cells. GSK3β silencing increased radiation-induced β-catenin target gene expression asmeasured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3β and β-catenin showed that GSK3β inhibition resulted in stabilization of β-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3β exhibited radioresistance in vivo. Silencing of β-catenin resulted in radiosensitization, whereas a nondegradable β-catenin construct induced radioresistance.
These data support the hypothesis that GSK3β modulates the cellular response to radiation in a β-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting β-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.
Pancreatic cancer is a deadly disease characterized by late diagnosis and resistance to therapy. Much progress has been made in defining gene defects in pancreatic cancer, but a full accounting of its molecular pathogenesis awaits. Here, we show that expression of Ataxia-Telangiectasia Group D Complementing gene (ATDC), also called TRIM29, is elevated in most invasive pancreatic cancers and pancreatic cancer precursor lesions. ATDC promoted cancer cell proliferation in vitro and enhanced tumor growth and metastasis in vivo. ATDC expression correlated with elevated β-catenin levels in pancreatic cancer, and β-catenin function was required for ATDC’s oncogenic effects. ATDC was found to stabilize β-catenin via ATDC-induced effects on the disheveled-2 protein, a negative regulator of GSK3β in the Wnt/β-catenin signaling pathway.
Pancreatic cancer is an aggressive malignancy, and an improved understanding of the molecular mechanisms governing its highly aggressive behavior is needed for more effective treatment, early detection and prevention. Defects in Wnt/β-catenin signaling are common in certain cancers, such as colorectal carcinoma, and recent evidence suggests Wnt/β-catenin signaling may contribute to pancreatic cancer. In this report, we show that ATDC is over-expressed in the majority of invasive pancreatic cancers and pancreatic cancer precursor lesions. ATDC contributes to pancreatic cancer via its ability to interact with and stabilize expression of disheveled-2, with resultant stabilization of β-catenin. Besides highlighting ATDC as a potential therapeutic target in pancreatic cancer, our studies have defined a novel mechanism for activating Wnt/β-catenin signaling in cancer.
pancreatic cancer; Wnt signaling; β-catenin; cell proliferation; disheveled
Dysregulation of β-catenin levels and localization and constitutive activation of β-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 β-catenin in the rat kidney epithelial cell line RK3E. We demonstrate expression of IRS1 can be directly activated by β-catenin, likely in part via β-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 β-catenin is an important regulator of IRS1 expression in vivo, we observed that IRS1 is highly expressed in many cancers with constitutive stabilization of β-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 β-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.
MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.
We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo.
Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells.
Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.
The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Here we show that different regions of the intracellular domain of DCC directly interacted with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activated both FAK and Src and stimulated tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduced DCC tyrosine phosphorylation and blocked both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolished its function of mediating netrin-induced axon attraction. On the basis of our observations, we suggest a model in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling.
Strategies for altering constitutional or somatic genotype in mice are well established, but approaches to generate mosaic genotypes in mouse tissues are limited. We showed that a functionally inactive Cre recombinase transgene with a long mononucleotide tract altering the reading frame was stochastically activated in the mouse intestinal tract. We demonstrated the utility of this approach by inducing colonic polyposis after Cre-mediated bi-allelic inactivation of the Apc gene.
A critical step in processing oligonucleotide microarray data is combining the information in multiple probes to produce a single number that best captures the expression level of a RNA transcript. Several systematic studies comparing multiple methods for array processing have used tightly controlled calibration data sets as the basis for comparison. Here we compare performances for seven processing methods using two data sets originally collected for disease profiling studies. An emphasis is placed on understanding sensitivity for detecting differentially expressed genes in terms of two key statistical determinants: test statistic variability for non-differentially expressed genes, and test statistic size for truly differentially expressed genes.
In the two data sets considered here, up to seven-fold variation across the processing methods was found in the number of genes detected at a given false discovery rate (FDR). The best performing methods called up to 90% of the same genes differentially expressed, had less variable test statistics under randomization, and had a greater number of large test statistics in the experimental data. Poor performance of one method was directly tied to a tendency to produce highly variable test statistic values under randomization. Based on an overall measure of performance, two of the seven methods (Dchip and a trimmed mean approach) are superior in the two data sets considered here. Two other methods (MAS5 and GCRMA-EB) are inferior, while results for the other three methods are mixed.
Choice of processing method has a major impact on differential expression analysis of microarray data. Previously reported performance analyses using tightly controlled calibration data sets are not highly consistent with results reported here using data from human tissue samples. Performance of array processing methods in disease profiling and other realistic biological studies should be given greater consideration when comparing Affymetrix processing methods.
Previous studies have demonstrated frequent allelic losses of distal chromosome 11q in ovarian carcinomas. The tumor suppressor gene(s) presumably targeted by these losses have not yet been identified. PPP2R1B is a candidate tumor suppressor gene at 11q23 that has recently been shown to be mutated in a subset of colorectal and lung cancers. We evaluated 5 ovarian carcinoma cell lines and 27 primary ovarian carcinomas for allelic losses of 11q23 and for mutations in the open reading frame of PPP2R1B. We also evaluated the primary tumors for allelic losses at 17p13, another chromosomal region frequently affected by losses of heterozygosity (LOH) in ovarian cancers. 11q23 and 17p13 allelic losses were identified in 25% and 74% of the carcinomas, respectively. No mutations within PPP2R1B coding sequences were found. These findings indicate that mutations of the PPP2R1B gene are infrequent in ovarian cancer and that deletions affecting the distal portion of chromosome 11q in ovarian cancer likely target inactivation of other genes.
tumor suppressor gene; human chromosome 11; loss of heterozygosity; ovarian carcinoma; PPP2R1B
Current models predict that β-catenin (β-cat) functions in Wnt signaling via activation of Tcf/Lef target genes and that its abundance is regulated by the adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β) proteins. In colon and other cancers, mutations in APC or presumptive GSK3β phosphorylation sites of β-cat are associated with constitutive activation of Tcf/Lef transcription. In spite of assumptions about its oncogenic potential, prior efforts to demonstrate that mutated β-cat will induce neoplastic transformation have yielded equivocal results. We report here that mutated, but not wild-type, β-cat proteins induced neoplastic transformation of RK3E, an adenovirus E1A-immortalized epithelial cell line. Analysis of the properties of mutant β-cat proteins and studies with a dominant negative Tcf-4 mutant indicated that the ability of β-cat to bind and activate Tcf/Lef factors is crucial for transformation. c-myc has recently been implicated as a critical Tcf-regulated target gene. However, c-myc was not consistently activated in β-cat-transformed RK3E cells, and a dominant negative c-Myc mutant protein failed to inhibit β-cat transformation. Our findings underscore the role of β-cat mutations and Tcf/Lef activation in cancer and illustrate a useful system for defining critical factors in β-cat transformation.
The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologues Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, we sought to define Siah-1 domains critical for regulation of DCC. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sina loss-of-function alleles, failed to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogated its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, we found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilized the Siah-1 protein. Siah-1 was found to oligomerize with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC.