APC Tumor Suppressor Gene Research Articles

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

β-Catenin Mutations: Insights into the APC Pathway and the Power of Genetics.

See related article by Sparks et al., [Cancer Res 1998;58:1130–4][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In the mid 1990s, the human genome project was well underway, but it would still be more than 5 years before the first draft of the human genome was published and more

Abstract 2684: Modeling a prototypical type I ovarian carcinoma in the mouse based on oviductal versus ovarian epithelial transformation: which is superior

Abstract Type I ovarian carcinomas are relatively indolent tumors that are nonetheless deadly if detected too late. Endometrioid carcinoma (EC) is a prototypical Type I ovarian cancer subtype. Existing mouse models of the disease based on transformation of the ovarian surface epithelium (OSE) take advantage of known EC genetic signatures but fail to capture fully the clinical disease progression and tumor phenotype. We previously described a mouse ovarian EC model in which the Apc and Pten tumor suppressor genes are conditionally deleted in the OSE via bursal injection of adenovirus expressing Cre recombinase (AdCre). We now report a new mouse model of EC in which the Ovgp1 (oviductal glycoprotein) promoter regulates expression of tamoxifen-inducible Cre recombinase specifically in the mouse oviductal epithelium (equivalent to human fallopian tube epithelium [FTE]), and not in the OSE or endometrium. Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice treated with tamoxifen (TAM) develop oviductal tumors over months rather than weeks. The oviductal EC morphology closely resembles that of human ovarian ECs, with overt gland formation throughout, whereas ovarian bursal injection of AdCre in Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice results in OSE-derived ECs that are poorly differentiated and include a prominent spindle cell component, consistent with our prior work. OSE-derived tumors progress rapidly, with none of the mice surviving beyond 14 weeks post-AdCre injection. Littermates with FTE-derived tumors survive much longer (>30 weeks post TAM). The slow progression and late (typically >16 weeks post TAM) metastasis to ovary and omentum associated with the FTE-derived tumors are more akin to the relatively indolent behavior characteristic of Type I ovarian carcinoma in humans. To further evaluate which of the two types of mouse tumors more closely resemble human ovarian tumors, we profiled gene expression in 6 FTE- and 5 OSE-derived Apc/Pten-deficient tumors, and compared the groups to each other and to data from 99 human ovarian carcinomas we analyzed previously (including 37 endometrioid, 41 serous, and 13 clear cell, and 8 mucinous carcinomas). Based on gene expression, the FTE-derived tumors are more correlated to every class of human ovarian tumors (p < 2 X10−6 in all 4 comparisons), with highest correlation to human ECs (particularly those with mutant CTNNB1) and mucinous carcinomas. This new model of EC, based on transformation of Mullerian epithelium rather than the OSE, will likely prove more suitable for translational applications, including pre-clinical testing of novel therapeutics for ovarian EC. Citation Format: Rong Wu, Yali Zhai, Rork Kuick, Paloma Garcia, Anum Naseem, Eric R. Fearon, kathleen R. Cho. Modeling a prototypical type I ovarian carcinoma in the mouse based on oviductal versus ovarian epithelial transformation: which is superior. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2684.

Abstract 4276: Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line

Abstract Introduction: We recently described overexpression of the enzyme, cystathionine-β-synthase (CBS) in human CRC (but not normal colonic mucosa) produces endogenous hydrogen sulfide (H2S) increasing tumor bioenergetics, cell proliferation, invasion, migration and promotes tumor angiogenesis. Its role in the progression of a colorectal adenoma to carcinoma remains elusive. The purpose of this study was to determine whether CBS overexpression in a non-tumorigenic human CRC cell line (NCM356) is sufficient to increase cell proliferation, migration, tumorigenesis, and metastasis. Methods: NCM356-p (parental) are non-tumorigenic when xenografted into athymic nude mice and express low levels of endogenous CBS, similar to normal colonic mucosa. RNASeq analysis determined mutation status. NCM-p were transduced with a lentiviral vector containing a CBS cDNA (NCM-C) or vector (NCM-v). H2S production was visualized with a fluorescent probe, 7-azido-4-methylcoumarine (AzMC). Cell proliferation rates where determined with a Coulter Counter. Cell migration and invasion assay were performed in Boyden chambers with NIH3T3 conditioned media (CM) as a chemoattractant. Anchorage-independent growth was assessed by soft-agar assay. Cellular bioenergetics was assessed using Oxygraph-O2K respirometer chamber. Tumorigenesis and metastasis were assessed injecting cells subcutaneously and orthotopically into nude mice, respectively. Aminooxyacetic acid (AOAA) was used to inhibit CBS activity. Statistical significance (p≤0.05) set using ANOVA or non-parametric Student t-test. Results: CBS overepression and H2S production was verified by Western blot and AzMC fluorescence in the NCM-C cells. CBS overexpression demonstrated significantly increased proliferation rate (p<0.03 NCM-C vs. NCM-v or NCM-p) over 4 days in culture, invasion through matrigel (p<0.01), migration (p<0.001) toward NIH3T3 CM and increased colony formation in soft agar (p<0.01). CBS inhibition with AOAA (1mM) decreased invasion and migration (p<0.01) in the NCM-C cells compared to NCM-v. In high-resolution respirometry, respiratory rate was significantly higher in the NCM-C compared to -p and -v. (141.8 ± 1.5 vs. 33.7 ± 0.8 vs. 72.2 ± 1.77 pmol·s−1·mg−1, -p v -C p < 0.0001, -v v -C p < 0.0005, -p v -v p < 0.001). Exome-wide sequence analyses of the NCM-p identified inactivating mutations in the APC and TP53 tumor suppressor genes, and an activating mutation in KRAS. Although the NCM-C cells produced significantly larger tumors in mice compared with NCM-v or NCM-p (p<0.001 2-w ANOVA), none of the cell lines caused liver metastasis in vivo. Conclusion: A non-tumorigenic cell line with some known mutations can progress to a tumorigenic, but not metastatic, phenotype with CBS overexpression and H2S production. Our data supports the importance of CBS/H2S axis in the adenoma to carcinoma sequence. Citation Format: John R. Zatarain, Ches’Nique M. Phillips, Michael E. Nicholls, Paul Johnson, Steven G. Widen, Thomas G. Wood, Nadiya Druzhyna, Bartosz Szczesny, Craig Porter, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4276.

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

Background and AimsColorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss.ResultsElevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated.MethodsHmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice.ConclusionsHMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.

Although human LINE-1 (L1) elements are actively mobilized in many cancers, a role for somatic L1 retrotransposition in tumor initiation has not been conclusively demonstrated. Here, we identify a novel somatic L1 insertion in the APC tumor suppressor gene that provided us with a unique opportunity to determine whether such insertions can actually initiate colorectal cancer (CRC), and if so, how this might occur. Our data support a model whereby a hot L1 source element on Chromosome 17 of the patient's genome evaded somatic repression in normal colon tissues and thereby initiated CRC by mutating the APC gene. This insertion worked together with a point mutation in the second APC allele to initiate tumorigenesis through the classic two-hit CRC pathway. We also show that L1 source profiles vary considerably depending on the ancestry of an individual, and that population-specific hot L1 elements represent a novel form of cancer risk.

Molecular Biomarkers in the Personalized Treatment of Colorectal Cancer.

Colorectal cancer (CRC) is a disease in which pathogenesis is influenced by genetic and epigenetic events that occur with tumor initiation and progression. Large variation exists in individual patient prognosis and response to chemotherapy, caused by molecular heterogeneity. Certain biomarkers have been identified that can predict clinical outcome beyond tumor staging, and inform treatment selection. Molecular testing is routinely performed in clinical practice for the selection of patients for targeted biological agents or immunotherapy, and is advocated for prognostic stratification. Estimating prognosis can avoid undertreatment or overtreatment and also guide the intensity of patient follow-up. Classifiers of CRC have been developed that integrate genetic and/or epigenetic features. The mutational status of KRAS and BRAF(V600E) oncogenes combined with analysis of the DNA mismatch repair system with/without the CpG island methylator phenotype (CIMP) has been shown to identify colon cancer subtypes with distinct clinical features and prognoses. Gene expression profiling has also been used to subtype CRCs and can overcome the limitations of single/limited gene testing. A recent effort identified 4 consensus molecular subtypes of biological relevance that were associated with different patient outcomes. Efforts to validate and refine these subtypes to include additional genomic features are ongoing. The focus of this article is to highlight molecular markers that can inform clinical decision-making in patients with CRC.

Hsa-miR-29c and hsa-miR-135b differential expression as potential biomarker of gastric carcinogenesis.

To investigate the expression profiles of hsa-miR-29c and hsa-miR-135b in gastric mucosal samples and their values as gastric carcinogenesis biomarkers. The expression levels of hsa-miR-29c and hsa-miR-135b in normal gastric mucosa, non-atrophic chronic gastritis, intestinal metaplasia and intestinal-type gastric adenocarcinoma were analysed using quantitative real-time PCR. The difference between hsa-miR-29c and hsa-miR-135b expression profiles in the grouped samples was evaluated by ANOVA and Student's t-test tests. The results were adjusted for multiple testing by using Bonferroni's correction. P values ≤ 0.05 were considered statistically significant. To evaluate hsa-miR-29c and hsa-miR-135b expressions as potential biomarkers of gastric carcinogenesis, we performed a receiver operating characteristic curve analysis and the derived area under the curve, and a Categorical Principal Components Analysis. In silico identification of the genetic targets of hsa-miR-29c and hsa-miR-135b was performed using different prediction tools, in order to identify possible genes involved in gastric carcinogenesis. The expression levels of hsa-miR-29c were higher in normal gastric mucosal samples, and decreased progressively in non-atrophic chronic gastritis samples, intestinal metaplasia samples and intestinal-type gastric adenocarcinoma samples. The expression of hsa-miR-29c in the gastric lesions showed that non-atrophic gastritis have an intermediate profile to gastric normal mucosa and intestinal-type gastric adenocarcinoma, and that intestinal metaplasia samples presented an expression pattern similar to that in intestinal-type gastric adenocarcinoma. This microRNA (miRNA) has a good discriminatory accuracy between normal gastric samples and (1) intestinal-type gastric adenocarcinoma; and (2) intestinal metaplasia, and regulates the DMNT3A oncogene. hsa-miR-135b is up-regulated in non-atrophic chronic gastritis and intestinal metaplasia samples and down-regulated in normal gastric mucosa and intestinal-type gastric adenocarcinoma samples. Non-atrophic chronic gastritis and intestinal metaplasia are significantly different from normal gastric mucosa samples. hsa-miR-135b expression presented a greater discriminatory accuracy between normal samples and gastric lesions. This miRNA was associated with Helicobacter pylori presence in non-atrophic chronic gastritis samples and regulates the APC and KLF4 tumour suppressor genes. Our results provide evidence of epigenetic alterations in non-atrophic chronic gastritis and intestinal metaplasia and suggest that hsa-miR-29c and hsa-miR-135b are promising biomarkers of gastric carcinogenesis.

New era of colorectal cancer screening.

Colorectal cancer (CRC) is the 2(nd) most common cancer in women and 3(rd) most common cancer in men worldwide. Most CRCs develop from adenomatous polyps arising from glandular epithelium. Tumor growth is initiated by mutation of the tumor suppressor gene APC and involves other genetic mutations in a stepwise process over years. Both hereditary and environmental factors contribute to the development of CRC. Screening has been proven to reduce the incidence of CRC. Screening has also contributed to the decrease in CRC mortality in the United States. However, CRC incidence and/or mortality remain on the rise in some parts of the world (Eastern Europe, Asia, and South America), likely due to factors including westernized diet, lifestyle, and lack of healthcare infrastructure. Multiple screening options are available, ranging from direct radiologic or endoscopic visualization tests that primarily detect premalignant or malignant lesions such as flexible sigmoidoscopy, optical colonoscopy, colon capsule endoscopy, computed tomographic colonography, and double contrast barium enema - to stool based tests which primarily detect cancers, including fecal DNA, fecal immunochemical test, and fecal occult blood test. The availability of some of these tests is limited to areas with high economic resources. This article will discuss CRC epidemiology, pathogenesis, risk factors, and screening modalities with a particular focus on new technologies.

Arid1a inactivation in an Apc- and Pten-defective mouse ovarian cancer model enhances epithelial differentiation and prolongs survival.

Inactivation of the ARID1A tumour suppressor gene is frequent in ovarian endometrioid (OEC) and clear cell (OCCC) carcinomas, often in conjunction with mutations activating the PI3K-AKT and/or canonical Wnt signalling pathways. Prior work has shown that conditional bi-allelic inactivation of the Apc and Pten tumour suppressor genes in the mouse ovarian surface epithelium (OSE) promotes outgrowth of tumours that reflect the biological behaviour and gene expression profiles of human OECs harbouring comparable Wnt and PI3K-AKT pathway defects, although the mouse tumours are more poorly differentiated than their human tumour counterparts. We found that conditional inactivation of one or both Arid1a alleles in OSE concurrently with Apc and Pten inactivation unexpectedly prolonged the survival of tumour-bearing mice and promoted striking epithelial differentiation of the cancer cells, resulting in morphological features akin to those in human OECs. Enhanced epithelial differentiation was linked to reduced expression of the mesenchymal markers N-cadherin and vimentin, and increased expression of the epithelial markers Crb3 and E-cadherin. Global gene expression profiling showed enrichment for genes associated with mesenchymal-epithelial transition in the Arid1a-deficient tumours. We also found that an activating (E545K) Pik3ca mutation, unlike Pten inactivation or Pik3ca H1047R mutation, cannot cooperate with Arid1a loss to promote ovarian cancer development in the mouse. Our results indicate that the Arid1a tumour suppressor gene has a key role in regulating OEC differentiation, and paradoxically the mouse cancers with more initiating tumour suppressor gene defects had a less aggressive phenotype than cancers arising from fewer gene alterations. Microarray data have been deposited in NCBI's Gene Expression Omnibus (GSE67695).

Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 receptor-insulin receptor substrate and STAT3 signaling.

The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and Signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer (CRC) development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (ApcMin/+) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Targeting JAK kinase in solid tumors: emerging opportunities and challenges

Various human malignancies are characterized by excessive activation of the Janus family of cytoplasmic tyrosine kinases (JAK) and their associated transcription factors STAT3 and STAT5. In the majority of solid tumors, this occurs in response to increased abundance of inflammatory cytokines in the tumor microenvironment prominently produced by infiltrating innate immune cells. Many of these cytokines share common receptor subunits and belong to the interleukin (IL)-6/IL-11, IL-10/IL-22 and IL-12/IL-23 families. Therapeutic inhibition of the JAK/STAT3 pathway potentially offers considerable benefit owing to the capacity of JAK/STAT3 signaling to promote cancer hallmarks in the tumor and its environment, including proliferation, survival, angiogenesis, tumor metabolism while suppressing antitumor immunity. This is further emphasized by the current successful clinical applications of JAK-specific small molecule inhibitors for the treatment of inflammatory disorders and hematopoietic malignancies. Here we review current preclinical applications for JAK inhibitors for the treatment of solid cancers in mice, with a focus on the most common malignancies emanating from oncogenic transformation of the epithelial mucosa in the stomach and colon. Emerging data with small molecule JAK-specific adenosine triphosphate-binding analogs corroborate genetic findings and suggest that interference with the JAK/STAT3 pathway may suppress the growth of the most common forms of sporadic colon cancers that arise from mutations of the APC tumor suppressor gene. Likewise inhibition of cytokine-dependent activation of the JAK/STAT3 pathway may also afford orthogonal treatment opportunities for other oncogene-addicted cancer cells that have gained drug resistance.

Expression and promoter methylation status of hMLH1, MGMT, APC, and CDH1 genes in patients with colon adenocarcinoma.

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor's formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient's age and tumor's clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.

Hunk/Mak-v is a negative regulator of intestinal cell proliferation.

BackgroundConditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer.MethodsHere we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis.ResultsWe show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis ApcMin mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival.ConclusionsIn the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1087-2) contains supplementary material, which is available to authorized users.

Detection of APC mosaicism by next-generation sequencing in an FAP patient.

Familial adenomatous polyposis (FAP) of the colon is characterized by multiple polyps in the intestine and extra-colonic manifestations. Most FAP cases are caused by a germline mutation in the tumor-suppressor gene APC, but some cases of adenomatous polyposis result from germline mutations in MUTYH, POLD1 or POLE. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic FAP patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. Whole-genome sequencing and subsequent deep sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in ~12% of his peripheral leukocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data implied that genetic analysis by next-generation sequencing is an effective strategy to identify genetic mosaicism in hereditary diseases.

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

Abstract PR08: Man's best friend for cancer driver-passenger discrimination

Abstract Spontaneously occurring canine cancer represents one of the best cancer models. Herein we report a novel dog human comparison strategy that utilizes inter-species genomic arrangements occurring during the past >75 million years to tackle a central aim of cancer research, namely cancer driver passenger discrimination. After showing that spontaneous canine colorectal cancers share similar molecular pathogenesis mechanisms as their human counterparts (Tang et al., 2010; Youmans et al., 2012), we compared copy number abnormalities between the two species (Tang et al., 2013). This led to the identification of 73 driver candidate genes (DCGs) and 38 passenger candidate genes (PCGs). Importantly, DCGs differ from PCGs in every analysis conducted to assess their cancer relevance. Moreover, while PCGs' functions appear random, DCGs' are significantly associated with cell proliferation and epithelial cell polarities. We are conducting 3D culture to validate these functions, leading to the discovery of several new tumor suppressors and cell polarity genes. Besides colorectal cancer, we have also successfully applied this dog-human comparison strategy on triple negative breast cancers, of which copy number abnormalities are extensive. In summary, we have developed a novel approach to efficiently analyze the massive cancer genomic data generated by TCGA and others for cancer driver discovery. Tang, J., Le, S., Sun, L., Yan, X., Zhang, M., Macleod, J., Leroy, B., Northrup, N., Ellis, A., Yeatman, T. J., et al. (2010). Copy number abnormalities in sporadic canine colorectal cancers. Genome research 20, 341-350. Tang, J., Li, Y., Lyon, K., Camps, J., Dalton, S., Ried, T., and Zhao, S. (2013). Cancer driver-passenger distinction via sporadic human and dog cancer comparison: a proof-of-principle study with colorectal cancer. Oncogene. Youmans, L., Taylor, C., Shin, E., Harrell, A., Ellis, A. E., Seguin, B., Ji, X., and Zhao, S. (2012). Frequent alteration of the tumor suppressor gene APC in sporadic canine colorectal tumors. PloS one 7, e50813. This abstract is also presented as Poster B51. Citation Format: Shaying Zhao. Man's best friend for cancer driver-passenger discrimination. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR08.

Early Detection of CHRPE and Subsequent Familial Adenomatous Polyposis Syndrome by Ultra-Wide Field Retinal Laser Scanning Technology

Introduction: Congenital hypertrophy of the retinal pigment epithelium (CHRPE) has been associated with familial adenomatous polyposis (FAP) and may be the first presenting feature of the disease process. This case presents the diagnosis of CHRPE by ultra-wide field retinal laser scanning technology. A 68-year-old male who was seen in the outpatient ophthalmologic care setting for post-op follow-up 1 month after photorefractive keratectomy (PRK) of OU in both eyes and correction of retinal detachment (RD) of the (OS) left eye. This patient has also previously tolerated phacoemulsification (PE) and intraocular lens placement (IOL), with open capsule for a cataract and Lasik surgery of OU without complication. Patient came in with complaints of his distance vision not being as clear as before the RD. Near is closer in the OS than before. Post-op follow up included ultra-wide field retinal laser scanning that reveal an OS peripheral CHRPE, and vitelliform dystrophy. We recommended IOL and referred the patient to oncology and gastrotroenterology, where he was diagnosed and treated as a new incident case of FAP. The most common and very first extracolonic manifestation is CHRPE, with reports of expression in 36-90% of FAP kindred, and with a specificity of 95% for being a carrier of the APC tumor suppressor gene deletion that leads to FAP. This tumor suppressor gene was identified at the long arm of chromosome 5 (5q21). Guidelines using older technology recommended that patients with more than 3 CHRPE in 1 eye or bilateral CHRPE, as well as patients with a positive family history and 1 unilateral CHRPE require further gastroenterological evaluation. In the future, the use of ultra-wide field retinal laser technology may detect CHRPE earlier than standard diagnostics, and subsequently lead to timely care for FAP.Figure 1

Abstract 417: DNA methylation changes in Lynch syndrome and FAP-associated colorectal tumorigenesis

Abstract Colorectal cancer (CRC) develops via multiple steps which involve genetic changes, such as mutations in growth-regulatory genes, and epigenetic alterations, such as CpG island hypermethylation. These changes accumulate over time in the normal tissue, for example due to many environmental factors such as diet, and may result in pre-cancerous field defects. Genes having high level of methylation in peritumoral tissues could be used as biomarkers for colorectal carcinogenesis. The most common familial cancer susceptibility syndromes are familial adenomatous polyposis (FAP), caused by inherited mutations in the APC tumor suppressor gene, and Lynch syndrome (LS), associated with inherited defects of the DNA mismatch repair (MMR) genes which together with other genetic and epigenetic changes are known to accelerate tumorigenesis. This investigation was designed to define how CpG island methylation of CRC risk genes varies in pre-cancerous and cancerous lesions, adjacent normal mucosa and distinct normal mucosa from genetically predisposed individuals. Colonic mucosa biopsies from different colorectal regions were gathered from 40 Finnish LS mutation carriers and 23 FAP patients during regular surveillance. In addition with normal mucosa biopsies, all adenoma and carcinoma findings from these individuals together with matching normals were gathered and analyzed. Patient information was obtained from the nation-wide registry. CpG island methylation was studied with the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) which detects CpG methylation of selected genes. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. Of 16 CRC risk genes tested so far 9 show increased methylation in polyps/adenomas/carcinomas. Mean methylation values increase along with age in both syndromes, with polyps from LS showing the highest proportional increase relative to normal tissues. The results offer new insights to the molecular mechanisms through which DNA methylation can affect colorectal carcinogenesis in LS and FAP mutation carriers. Citation Format: Satu Valo, Sippy Kaur, Ari Ristimäki, Laura Renkonen-Sinisalo, Heikki Järvinen, Jukka-Pekka Mecklin, Minna Nyström, Päivi Peltomäki. DNA methylation changes in Lynch syndrome and FAP-associated colorectal tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2014-417