Loss Of Apc Research Articles

Abstract A02: High-depth sequencing reveals the presence of multi-clonality in colorectal premalignancy

Abstract Clonality in carcinomas is thought to arise from the accumulation of mutations and changes in selective pressure during carcinogenesis and further stages of tumor development. However, the identification of clonality through different molecular and systems biology tools has challenged the current understanding of carcinogenesis and progression. Familial Adenomatous Polyposis (FAP) is a genetic condition secondary to germline mutations in APC and characterized by the development of hundreds of polyps in the colorectum. Therefore, FAP is an ideal model to study the presence of clonality during early stages of colorectal carcinogenesis in humans. Second hits in APC are a valuable and intuitive method to establish the presence of clonality in FAP, as additional somatic APC mutations will reflect the presence of additional clones. Here, we present a targeted high-depth sequencing analysis of APC and KRAS in conjunction with high density SNP array data in 37 colorectal adenomas and matched adjacent uninvolved mucosa samples. Ampliseq sequencing was performed using the next-generation sequencing platform IT Personal Genome Machine (PGM; Life Technologies) using the Ion PGM 200 Sequencing Kit on an Ion 318 Chip Kit (Life Technologies, average of 70% bases at 500x). Ion Torrent Variant Caller v4.2 was run in the somatic low stringency proton mode to detect variants in APC and KRAS against hg19. Then, normal variants were subtracted from matched adenoma variants to create a list of somatic candidates for each adenoma and normal pair. SNP array analysis was performed using the HumanOmniExpressExome-8 v1.2 BeadChip (Illumina, San Diego, CA). To validate the presence of double somatic hits in APC we performed a colony genotyping assay from one adenoma sample. First, we identified APC somatic mutations or loss of heterogeneity (LOH) in 5q (second hits) in 81% (30/37) and KRAS somatic mutations in 35% (16/37) of adenomas analyzed. Six adenomas (16%) presented double somatic APC mutations with different allelic frequencies with three of them showing a significant difference in allelic frequencies among the somatic APC hits (>30%); in addition, one adenoma presented two KRAS mutations. The APC clonality was validated in one adenoma using colony analysis. The region that contains the two somatic mutations (c.4348C>T and c.4267_4280del) and the germline mutation (c.2894delA) was amplified by Long-Range PCR and cloned into the pGEM-T plasmid. DNA from one hundred colonies was extracted and genotyped using Taqman assay probes. All samples were genotyped in triplicate and positive and negative controls for all mutations were included in every single plate. A total of 25% of the colonies harbored a wild-type allele, 36% the known germline APC mutation, 15% the APC somatic c.4348C>T mutation and 24% the APC somatic c.4267_4280del mutation. In conclusion, our analysis showed that there are multiple mutated APC subclones driving initiation of colorectal carcinogenesis, thus supporting experimentally a finding of multi-clonality in premalignant colonic lesions. These findings support a model of colorectal carcinogenesis that is based on polycryptal polyclonal adenomas and calls for revisiting the “Big-Bang” model based on the presence of multiple initiator clones. Further analyses aimed to understand the functional impact and the hierarchical relations among the different identified subclones are warranted. Citation Format: Ester Borras, Kyle Chang, Anthony San Lucas, Gita Masand, Axel Delgado Amador, Jerry Fowler, Gareth E. Davies, Erik A. Ehli, Y. Nancy You, Patrick M. Lynch, Ernest T. Hawk, Gabriel Capella, Paul A. Scheet, Eduardo Vilar. High-depth sequencing reveals the presence of multi-clonality in colorectal premalignancy. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A02.

Suppression of intestinal tumorigenesis in Apc mutant mice upon Musashi-1 deletion.

Therapeutic strategies based on a specific oncogenic target are better justified when elimination of that particular oncogene reduces tumorigenesis in a model organism. One such oncogene, Musashi-1 (Msi-1), regulates translation of target mRNAs and is implicated in promoting tumorigenesis in the colon and other tissues. Msi-1 targets include the tumor suppressor adenomatous polyposis coli (Apc), a Wnt pathway antagonist lost in ∼80% of all colorectal cancers. Cell culture experiments have established that Msi-1 is a Wnt target, thus positioning Msi-1 and Apc as mutual antagonists in a mutually repressive feedback loop. Here, we report that intestines from mice lacking Msi-1 display aberrant Apc and Msi-1 mutually repressive feedback, reduced Wnt and Notch signaling, decreased proliferation, and changes in stem cell populations, features predicted to suppress tumorigenesis. Indeed, mice with germline Apc mutations (ApcMin ) or with the Apc1322T truncation mutation have a dramatic reduction in intestinal polyp number when Msi-1 is deleted. Taken together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis driven by Apc loss, and validate the pursuit of Msi-1 inhibitors as chemo-prevention agents to reduce tumor burden.

HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation

Cancer genome sequencing projects have identified hundreds of genetic alterations, often at low frequencies, raising questions as to their functional relevance. One exemplar gene is HUWE1, which has been found to be mutated in numerous studies. However, due to the large size of this gene and a lack of functional analysis of identified mutations, their significance to carcinogenesis is unclear. To determine the importance of HUWE1, we chose to examine its function in colorectal cancer, where it is mutated in up to 15 per cent of tumours. Modelling of identified mutations showed that they inactivate the E3 ubiquitin ligase activity of HUWE1. Genetic deletion of Huwe1 rapidly accelerated tumourigenic in mice carrying loss of the intestinal tumour suppressor gene Apc, with a dramatic increase in tumour initiation. Mechanistically, this phenotype was driven by increased MYC and rapid DNA damage accumulation leading to loss of the second copy of Apc. The increased levels of DNA damage sensitised Huwe1‐deficient tumours to DNA‐damaging agents and to deletion of the anti‐apoptotic protein MCL1. Taken together, these data identify HUWE1 as a bona fide tumour suppressor gene in the intestinal epithelium and suggest a potential vulnerability of HUWE1‐mutated tumours to DNA‐damaging agents and inhibitors of anti‐apoptotic proteins.

Aggressive Aneuploid Acute Myeloid Leukemia Is Dependent on Alterations of P53, Gain of APC and PLK1 and Loss of RAD50

Abstract Chromosome number alterations, aneuploidy, is a hallmark of cancer. It occurs in about 15% of acute myeloid leukemia (AML) cases, is generally preserved throughout disease progression (Bochtler et al. Leukemia 2015) and correlates with adverse prognosis (Breems et al. JCO 2008, Papaemmanuil et al. NEJM 2016). This evidence highlights the need of understanding the molecular mechanisms that promote and sustain aneuploidy in AML, in order to define novel potential therapeutic targets. In the NGS-PTL project we profiled the genomic landscape of 536 hematological samples by whole exome sequencing (WES, Illumina). Among them, we analyzed 88 and 68 samples from aneuploid (A-) FLT3-wildtype AML (isolated trisomy and monosomy, complex and monosomal karyotype) and euploid (E-) AML (normal and complex karyotype, <3 abnormalities), respectively (100 bp, paired-end). Variants were called by MuTect and Varscan 2.0. WES output was integrated with genotype data (CytoScan HD Array, Affymetrix and Nexus Copy Number analysis) and gene expression profiling (HTA 2.0 and TAC 3.0, Affymetrix). A-AML showed an increased genomic instability, as confirmed by a higher mutation load compared with E-AML (median number of variants: 22 (range: 2-95) and 11 (range: 3-45), respectively, p<.001), which was associated with increased patients' age (median age of 62 for A-AML and 55 for E-AML, p<.05). The increased age and mutation load correlated with a mutational signature with prominence of C>A substitutions, compared with the C>T transition-related signature, which is prevalent in AML. A-AML was associated with mutations and/or heterozygous deletion of TP53 (p<.001), which co-occurred with copy number loss of both the tumor suppressor APC and the DNA repair gene RAD50 in 93% of cases (p<.001). Moreover, A-AML was enriched for a gene expression signature of p53-deficiency, independently of TP53 structural defects (p<.05, GSEA). Mutations and deregulated expression of genes involved in cell cycle contributed to the A-AML phenotype, with 68% A-AML vs. 32% E-AML patients (p<.01) carrying at least one genomic lesion affecting the process. The alterations targeted the following pathways: DNA repair (i.e. reduced RAD50 expression, p<.001), cell cycle checkpoints (i.e. mutated CHK2), regulation of PLK1 activity at G2/M transition (i.e. mutation and 2-fold upregulation of PLK1, p<.01), mitotic metaphase and anaphase (i.e. increased CDC20 level, p<.001) and separation of sister chromatids (i.e. mutated BUB1B, ESPL1, CENPO). Of note, a 3-gene signature composed of PLK1, CDC20 and RAD50, was able to discriminate 73% of patients between the A- and E-AML cohorts. This signature was confirmed at protein level. In parallel, E-AML showed a preferential dependency on epigenetic mechanisms, with recurrent genomic lesions of ASXL1/2, BCOR/L1, EZH2 and MLL, enrichment of FLT3 alterations and mutations activating RAS signal transduction (p<.05). Of note, a HOX-related signature characterized by overexpression of the HOX family members HOXA7, HOXB3 and MEIS1 identified E-AML. We show here for the first time the molecular mechanisms promoting and maintaining aneuploidy in AML. Our results indicate that p53 deficiency, either caused by somatic mutations, copy number loss, impaired DNA damage response and enhanced PLK1 signaling synergize with APCgain, RAD50 structural or functional loss and forced progression through mitosis, to override cell cycle and mitotic checkpoints and allow the formation of daughter cells with an aberrant chromosome number. These mechanisms cooperate with recurrent mutations of genes involved in protein ubiquitination and proteasome-mediated protein catabolic process in A-AML, indicative of the attempt of aneuploid cells to override the proteotoxic stress due to the unbalanced protein load generated by the aneuploid condition. This evidence provides the rationale for exploiting proteasome inhibition (Velcade), p53 reactivation (MDM2/4 inhibitor) and targeting of the cell cycle (CHK1/2 inhibitor) downstream to p53 (WEE1 inhibitor) as strategies for novel combination therapies against aggressive aneuploid AML, which are under clinical investigation in our Institution and may serve as a model for aneuploid tumors. GS and AP: equal contribution. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Novartis: Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Inhibition of Canonical WNT Signaling in the Bone Marrow Niche Prevents the Development of Disease in a Mouse MDS Model with Apc Haploinsufficiency

The myelodysplastic syndromes (MDS) include a spectrum of clonal hematopoietic stem cell (HSC) disorders that are characterized by ineffective hematopoiesis, peripheral cytopenia(s), morphologic dysplasia, and a variable propensity to transform to acute myeloid leukemia (AML). A del(5q) is the most common recurring cytogenetic abnormality in MDS, and is observed in 10-20% of primary MDS, and 40% of therapy-related MDS and AML. Our lab has had a long-standing interest in understanding how loss of function of tumor suppressor genes on 5q, such as APC, contributes to the pathogenesis of MDS and AML.Apc is a key negative regulator of the canonical β-catenin (Ctnnb1)/WNT signaling pathway.WNT signaling has been implicated in self-renewal of HSCs and the regulation of hematopoiesis, and the deregulation of WNT signaling is associated with the development of hematological malignancies. Recent data suggests that WNT activation in the bone marrow (BM) microenvironment or niche may also contribute to the development of AML. Kode et al. (Nature 506:240, 2014) showed that expression of activated β-catenin in osteoblast cells in the BM microenvironment leads to AML in mice. Thus, aberrant WNT signaling in HSCs and/or the microenvironment that supports them may contribute to malignant transformation. We previously used a conditional mouse model (Mx1-Cre+, Apc fl/+, referred to as Apcdel/+) and showed that haploinsufficiency of Apc leads to ineffective hematopoiesis (Blood 115:3481, 2010). Interestingly, haploinsufficient loss of Apc in the niche stromal cells, but not the hematopoietic progenitors, led to the development of MDS, characterized by a severe macrocytic anemia, dyserythropoiesis, megakaryocytic proliferation, and mild granulocytic dysplasia (Blood 123:228, 2014). In this study, we sought to determine whether haploinsufficient loss of Apc, mediates this disease through modulating WNT signaling, versus other Apc functions, and whether inhibition of WNT signaling could prevent the development of MDS in Apcdel/+mice.Here, we demonstrate thatsignaling through the canonical WNT pathway mediates the development of MDS in Apc haploinsufficient mice. Specifically, loss of one copy of Ctnnb1 is sufficient to prevent the development of MDS in Apcdel/+ mice. Compared to the median survival of Apc del/+ mice, which was 255 days, all Apc del/+, Ctnnb1del/+ mice survived until the end of the study (~400 days) (P<0.001). In addition, we showed that altered canonical WNT signaling in the BM microenvironment is responsible for the disease. Using bone marrow transplantation of wild type BM cells, we showed that Apc del/+ recipients develop MDS and die of a fatal anemia by ~4 months of age. In contrast, Apc del/+, Ctnnb1 del/+ recipients survived on average 8-10 months longer (median survival 115 vs 413 days, P <0.0001). To determine if we could prevent the development of MDS, we treated mice with the FDA-approved antihelminth drug, Pyrvinium, an inhibitor of the WNT pathway via activation of casein kinase 1 alpha and β-catenin degradation. Apc del/+mice in the control DMSO-treated group developed disease with a median survival of 227 days; however, during this time period, none of the Pyrvinium-treated mice became moribund. The mice succumbed to MDS about 100 days after Pyrvinium treatment was suspended, indicating that the drug prevented the development of disease, and must be continuously administered. We also showed that Pyrvinium treatment does not have to be administered before anemia develops; however, the drug is more effective when it is started in animals that display mild-to-moderate anemia rather than severe anemia.These studies have important implications for the treatment of certain forms of MDS. Kode et al. (Nature 506:240, 2014) showed nuclear accumulation of β-catenin in osteoblasts from ~40% of MDS/AML patients, supporting the idea that activated WNT signaling in the BM microenvironment may contribute to the development of disease. Targeting the tumor microenvironment is now recognized as a promising approach to complement current therapies, and our data highlight a potential new strategy of targeting the WNT signaling pathway for treating some forms of MDS. DisclosuresNo relevant conflicts of interest to declare.

The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

BackgroundMost cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs.MethodsTwo FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed.ResultsWe have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population.ConclusionsOur results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2809-9) contains supplementary material, which is available to authorized users.

Gasdermin C Is Upregulated by Inactivation of Transforming Growth Factor β Receptor Type II in the Presence of Mutated Apc, Promoting Colorectal Cancer Proliferation.

Mutations in TGFBR2, a component of the transforming growth factor (TGF)-β signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-β-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-β-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Thrombomodulin‐dependent protein C activation is required for mitochondrial function and myelination in the central nervous system

Background Studies with human samples and in rodents established a function of coagulation proteases in neuro-inflammatory demyelinating diseases (e.g. in multiple sclerosis [MS] and experimental autoimmune encephalitis [EAE]). Surprisingly, approaches to increase activated protein C (aPC) plasma levels as well as antibody-mediated inhibition of PC/aPC ameliorated EAE in mice. Hence, the role of aPC generation in demyelinating diseases and potential mechanisms involved remain controversial. Furthermore, it is not known whether loss of aPC has pathological consequences at baseline (e.g. in the absence of disease). Objective To explore the role of thrombomodulin (TM)-dependent aPC generation at baseline and in immunological and non-immunological demyelinating disease models. Methods Myelination and reactive oxygen species (ROS) generation were evaluated in mice with genetically reduced TM-mediated protein C activation (TMPro/Pro ) and in wild-type (WT) mice under control conditions or following induction of EAE. Non-immunological demyelination was analyzed in the cuprizone-diet model. Results Impaired TM-dependent aPC generation already disturbs myelination and mitochondrial function at baseline. This basal phenotype is linked with increased mitochondrial ROS and aggravates EAE. Reducing mitochondrial ROS (p66Shc deficiency), restoring aPC plasma levels or injecting soluble TM (solulin) ameliorates EAE in TMPro/Pro mice. Soluble TM additionally conveyed protection in WT-EAE mice. Furthermore, soluble TM dampened demyelination in the cuprizone-diet model, demonstrating that its myelin-protective effect is partially independent of an immune-driven process. Conclusion These results uncover a novel physiological function of TM-dependent aPC generation within the CNS. Loss of TM-dependent aPC generation causes a neurological defect in healthy mice and aggravates EAE, which can be therapeutically corrected.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

Abstract 385: mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer

Abstract BACKGROUND Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States. Several key mutations in CRC include APC (80%), TP53 (50%), and PIK3CA (20-30%). Mutations in the PIK3CA gene, resulting in a constitutively active PI3K, often occur concomitantly with loss of the APC gene in human CRCs. Our lab has developed a murine model system where a constitutively active PI3K and loss of APC occur simultaneously in the colon (AK3K) as well as concomitant loss of p53 (AK3KTO). Colon tumors from these models are cultured as three-dimensional spheroids and treatment studies. METHODS AK3K and AK3KTO spheroids were treated with a dual PI3K/mTOR inhibitor, NVP-BEZ235, or a mTORC1/2 inhibitor, MLN0128. Images were taken both pre- and post-treatment and changes in spheroid diameter were measured. Parallel treatment studies were performed on a primary human colon cancer tumor cell line, SW48, which carries a mutant FBXW7 and also on a cell line transfectedwith a PIK3CA mutation (SW48PK). RESULTS Treatment with NVP-BEZ235 and MLN0128 resulted in a significant treatment response as measured by marked change in spheroid diameter in the AK3K spheroids at 100, 200 and 400nM (p&amp;lt;0.01), with MLN0128 eliciting a greater treatment response. There were no significant differences between drugs in the AK3KTO spheroids. Comparing across cell lines, there were highly significant differences when treated with NVP-BEZ235, as the treatment was much more effective in the AK3KTO cell line at 100, 200, and 400nM (p&amp;lt;0.01). There were no significant differences between cell lines for MLN0128. Immunoblotting further demonstrated a dramatic inhibition of the PI3K/mTOR pathway in both spheroids following treatment with 200nM MLN0128 and 200nM NVP-BEZ235. Treatment of SW48 and SW48PK cells with NVP-BEZ235 and MLN0128 showed highly significant differences between the drugs in both cell lines, with MLN0128 being more effective at 1, 10, and 50nM (p&amp;lt;0.01). At higher drug concentrations (100, 200, and 400nM), there were highly significant differences between drugs for the SW48PK cell line (p&amp;lt;0.01) but no significant differences in the SW48 cells. Comparing across cell lines, there were highly significant differences when treated with MLN0128 at 100, 200, and 400nM (p&amp;lt;0.01), with MLN0128 being more effective in the SW48PK cell line. Immunoblotting demonstrated a reduction in activation of the PI3K/mTOR pathway following treatment with 10nM MLN0128 but this inhibition was not seen when treated with 10nM NVP-BEZ235. CONCLUSIONS mTOR inhibition is sufficient to induce responses in PIK3CA and APC mutated CRCs and concomitant loss of TP53 does not lead to resistance and potentially increases sensitivity. In a cell line predisposed to PI3K/mTOR signaling sensitivity, mTOR inhibition remains sufficient and the addition of a PIK3CA mutation further increases sensitivity Citation Format: Stephanie Fricke, Cheri Pasch, Susan Payne, Alexander Yueh, Tyler Foley, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. mTORC1/2 inhibition as a treatment strategy for subtypes of colorectal cancer. [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 385.

Abstract 912: Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon

Abstract Intestinal crypts are maintained by long-lived intestinal stem cells (ISCs) which reside near the base of glands. Above the ISC pool, there are short-lived progenitors that can supply lineage-specific differentiated cell types into the villus. Notch and Wnt pathways play a key role for determining the stem/progenitor cell function and their cell fate, and regulating cancer development in the gastrointestine. Although it has been well established that ISCs are a major origin of intestinal cancer, it remains unknown whether intestinal progenitor population can give rise to cancer. We use Mist1-CreERT mice and induce Notch and Wnt activation by mating with LSL-Notch1-IC mice and/or Apc flox mice. We found that a bHLH transcriptional factor Mist1 is expressed in Paneth cells (Lysozyme+CD24high) as well as short-lived secretory progenitors (Lysozyme-CD24low) in the intestine. Mist1+ secretory progenitors are radio- and chemo-resistant, but do not show stem cell interconversion even after intestinal injury or Lgr5 ablation. However, aberrant Notch activation changes Mist1+ cells to Lgr5+ long-lived enterocyte progenitors, with loss of secretory lineage differentiation. Mist1+CD24low progenitor population can form intestinal organoids with Notch activation in vitro. Mist1+ secretory progenitors can give rise to intestinal cancer by simultaneous Notch and Wnt activation by loss of Apc gene, while loss of Apc in Mist1 lineage alone does not develop cancer. In the colon, Mist1 marks colonic secterory progenitors that become to lineage trace and can be a cancer-initiating cell after Notch activation or DSS-induced colonic injury. These results provide the clear evidence of cellular plasticity dependent on Notch signaling in the gut, and suggest short-lived progenitors as another cellular origin of cancer besides ISC pool. Citation Format: Yoku Hayakawa, Kosuke Sakitani, Woosook Kim, Yagnesh Tailor, Karan Nagar, Kazuhiko Koike, Samuel Asfaha, Timothy C. Wang. Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon. [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 912.

Abstract 2884: Role of Kaiso in intestinal tumorigenesis

Abstract p120-catenin is a master regulator of classical cadherin stability and essential for epithelial homeostasis. p120 physically interacts with transcription factor Kaiso (ZBTB33) suggesting a direct or indirect role in transcription, but its precise function with respect to Kaiso is largely unknown. Kaiso belongs to the ZBTB family of transcription factors, most of which have important roles in development and/or cancer. Interestingly, Kaiso is strongly expressed in the intestinal crypt and abruptly downregulated as the cells move up onto the villus and terminally differentiate. Kaiso is constitutively upregulated in nascent adenomas initiated by loss of the tumor suppressor APC, suggesting a link between canonical Wnt signaling, Kaiso upregulation and intestinal tumorigenesis. Here, using ChipSeq and proteomics we describe several candidate Kaiso interactions with a group of proteins that include the tumor suppressor BRCA1, CHD2 (a chromatin remodifier) and the Ets1 oncoprotein, along with preliminary evidence for functional significance relevant to intestinal tumorigenesis. Citation Format: Manish K. Tripathi, Albert B. Reynolds. Role of Kaiso in intestinal tumorigenesis. [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 2884.

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.

The Prosurvival IKK-Related Kinase IKKε Integrates LPS and IL17A Signaling Cascades to Promote Wnt-Dependent Tumor Development in the Intestine.

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkε in Wnt-driven tumor development. We found that Ikkε was activated in intestinal tumors forming upon loss of the tumor suppressor Apc Genetic ablation of Ikkε in β-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkε to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkε was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkε-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkε phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkε to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587-99. ©2016 AACR.

803 MIST1 Positive Stem Cells in the Antrum Serve As a Cell-of-Origin for Gastric Cancer With APC Loss

803 MIST1 Positive Stem Cells in the Antrum Serve As a Cell-of-Origin for Gastric Cancer With APC Loss

Bcl-2 is a critical mediator of intestinal transformation.

Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target.

High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (non-ISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoid-initiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how diet-modulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors.

Tumor promotion and inhibition by phenobarbital in livers of conditional Apc-deficient mice.

Activation of Wnt/β-catenin signaling is important for human and rodent hepatocarcinogenesis. In mice, the tumor promoter phenobarbital (PB) selects for hepatocellular tumors with activating β-catenin mutations via constitutive androstane receptor activation. PB-dependent tumor promotion was studied in mice with genetic inactivation of Apc, a negative regulator of β-catenin, to circumvent the problem of randomly induced mutations by chemical initiators and to allow monitoring of PB- and Wnt/β-catenin-dependent tumorigenesis in the absence of unknown genomic alterations. Moreover, the study was designed to investigate PB-induced proliferation of liver cells with activated β-catenin. PB treatment provided Apc-deficient hepatocytes with only a minor proliferative advantage, and additional connexin 32 deficiency did not affect the proliferative response. PB significantly promoted the outgrowth of Apc-deficient hepatocellular adenoma (HCA), but simultaneously inhibited the formation of Apc-deficient hepatocellular carcinoma (HCC). The probability of tumor promotion by PB was calculated to be much lower for hepatocytes with loss of Apc, as compared to mutational β-catenin activation. Comprehensive transcriptomic and phosphoproteomic characterization of HCA and HCC revealed molecular details of the two tumor types. HCC were characterized by a loss of differentiated hepatocellular gene expression, enhanced proliferative signaling, and massive over-activation of Wnt/β-catenin signaling. In conclusion, PB exerts a dual role in liver tumor formation by promoting the growth of HCA but inhibiting the growth of HCC. Data demonstrate that one and the same compound can produce opposite effects on hepatocarcinogenesis, depending on context, highlighting the necessity to develop a more differentiated view on the tumorigenicity of this model compound.

Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.