Loss Of Adenomatous Polyposis Coli Research Articles

Tumor Establishment Requires Tumor Autonomous and Non-Autonomous Decoupling of EGF Signaling from Apoptosis

During organ turnover, robust mechanisms enforce a balanced equilibrium between cell division and death. Nascent tumors must destabilize these mechanisms to subvert cell equilibrium for cancerous growth. The nature of these destabilizing events is central to understanding how new tumors become established and may suggest strategies for cancer prevention. One of the best characterized mechanisms for cell equilibrium is that of the adult Drosophila intestine. In this organ, division is coupled to death because Rhomboid, a protease that cleaves mitogenic EGFs for secretion, is induced in cells undergoing apoptotic elimination. In Drosophila, as in mammals, intestinal stem cells give rise to adenomas following loss of adenomatous polyposis coli (APC). Examining Drosophila APC-/- tumorigenesis, we find that pre-tumor cells destabilize cell equilibrium by uncoupling rhomboid from apoptosis, which creates feed-forward amplification of EGF signaling for tumor establishment. Prior to tumor formation, APC-/- cells induce rhomboid in surrounding cells via activation of the stress signal JNK. During subsequent growth, APC-/- tumors induce rhomboid within the tumor itself via loss of E-cadherin and consequent activity of p120-catenin. Chronic induction of rhomboid in both tumor and surrounding cells leads to constitutive activation of EGFR and is essential for tumors to progress. Thus, incipient tumors combine non-autonomous and autonomous strategies to deregulate rhomboid and destabilize cell equilibrium. Since Rhomboid, EGFR, and E cadherin are associated with colorectal cancer in humans, our findings may shed light on how human colorectal tumors progress.

Inhibition of phosphodiesterase 4D decreases the malignant properties of DLD-1 colorectal cancer cells by repressing the AKT/mTOR/Myc signaling pathway.

Colorectal cancer (CRC) is a complex disease involving numerous genetic abnormalities. One of the major characteristics of CRC is enhanced Wnt signaling caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene. Previously, it has been demonstrated that the majority of malignant phenotypes following APC deletion in adult murine small intestines could be rescued when Myc, a downstream target of the Wnt pathway, was deleted. This indicated that Myc is a critical regulator of CRC development following APC loss. Previous studies reported that cyclic adenosine 3',5'-monophosphate (cAMP) can influence the AKT/mammalian target of rapamycin (mTOR) survival pathway in cancer and Myc is a critical downstream molecule of AKT/mTOR signaling. Phosphodiesterase 4D (PDE4D), a member of the cAMP-specific PDE4 family, has been associated with drug resistance in CRC. However, the association between PDE4D and Myc remains unclear. To investigate the potential role of PDE4D in Myc regulation in CRC, the present study evaluated the expression levels of PDE4 subtypes in DLD-1 CRC cells. Additionally, the effects of PDE4 inhibitors on Myc expression and oncogenic properties were analyzed by western blot analysis, reverse transcription-quantitative polymerase chain reaction, colony formation and soft agar assays. It was demonstrated that cAMP/PDE4D signals serve a critical role in regulating Myc expression in DLD-1 CRC cells. Furthermore, PDE4D was identified to be a main hydrolyzer of cAMP and suppression of PDE4D using selective inhibitors of PDE4 increased intracellular cAMP levels, which resulted in a marked decrease in the oncogenic properties of DLD-1 cells, including colony formation, cell proliferation and anchorage-independent growth. Notably, the current data imply that cAMP represses Myc expression via the downregulation of AKT/mTOR signaling, which was abolished by high PDE4D activities in DLD-1 cells. Additionally, a natural polyphenol resveratrol in combination with forskolin elevated the concentration of cAMP and enhanced the expression of Myc and the malignant phenotype of DLD-1 cells, reproducing the effect of known chemical inhibitors of PDE4. In conclusion, the present study identified that cAMP/PDE4D signaling is a critical regulator of Myc expression in DLD-1 and possibly other CRC cells.

A small molecule approach to degrade RAS with EGFR repression is a potential therapy for KRAS mutation-driven colorectal cancer resistance to cetuximab

Drugs targeting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, have been prescribed for metastatic colorectal cancer (CRC), but patients harboring KRAS mutations are insensitive to them and do not have an alternative drug to overcome the problem. The levels of β-catenin, EGFR, and RAS, especially mutant KRAS, are increased in CRC patient tissues due to mutations of adenomatous polyposis coli (APC), which occur in 90% of human CRCs. The increases in these proteins by APC loss synergistically promote tumorigenesis. Therefore, we tested KYA1797K, a recently identified small molecule that degrades both β-catenin and Ras via GSK3β activation, and its capability to suppress the cetuximab resistance of KRAS-mutated CRC cells. KYA1797K suppressed the growth of tumor xenografts induced by CRC cells as well as tumor organoids derived from CRC patients having both APC and KRAS mutations. Lowering the levels of both β-catenin and RAS as well as EGFR via targeting the Wnt/β-catenin pathway is a therapeutic strategy for controlling CRC and other types of cancer with aberrantly activated the Wnt/β-catenin and EGFR-RAS pathways, including those with resistance to EGFR-targeting drugs attributed to KRAS mutations.

Microtubule plus-end tracking Adenopolyposis Coli negatively regulates proplatelet formation

Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.

MicroRNA-552 links Wnt signaling to p53 tumor suppressor in colorectal cancer.

The aberrant expression of microRNAs (miRNAs or miRs) has been shown to be associated with the development of various types of cancer, including colorectal cancer (CRC). The increased activation of the Wnt signaling pathway via the loss of the Wnt repressor, adenomatous polyposis coli (APC), is the hallmark of human CRC. In this study, we demonstrate that the activation of the Wnt/c-Myc axis inhibits the expression of the tumor suppressor, p53, via promoting the targeting of p53 by miR‑552. Our results revealed that the ectopic expression of miR‑552 enhanced cell proliferation, colony formation and resistance to drug-induced apoptosis, suggesting that this miRNA may function as an oncogene. We found that miR‑552 displayed oncogenic properties by directly targeting the p53 tumor suppressor. Of note, our genetic and pharmacological experiments revealed that the Wnt/β-catenin signaling pathway and its major downstream target, c-Myc (hereafter termed Myc), increased the miR‑552 levels, and chromatin immunoprecipitation (ChIP) assays revealed they carried out this function by directly binding to their binding sites in the miR‑552 promoter region. Given that the functional loss of APC, leading to abnormal Wnt signals, and the absence of p53 protein are common in CRC, these results suggest that miR‑552 may serve as an important link between these two events, and this warrants further investigation. Collectively, the data of this study suggest that the inhibition of miR‑552 may disconnect elevated Wnt signals from p53 suppression, providing a novel therapeutic strategy for patients with CRC with deregulated Wnt signaling.

Abstract 554: APC loss alters DNA damage repair in breast cancer cells

Abstract Breast cancer is the second leading cause of cancer deaths in U.S. women. Many of these deaths have been attributed to the development of chemotherapeutic resistance. Thus, understanding the process of chemotherapeutic resistance will help develop new approaches to combat breast cancer and ultimately cause a decrease in breast cancer related deaths. The Adenomatous Polyposis Coli (APC) tumor suppressor is silenced or mutated in up to 70% of sporadic breast cancer; however, the possible effects of APC loss on chemoresistance has not been elucidated. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we previously showed that APC loss decreased cisplatin and doxorubicin-induced apoptosis. We also have shown that APC loss increased mRNA expression of the multidrug resistance protein 1 (MDR1), which was augmented by doxorubicin treatment. Treatment with doxorubicin and A69, a STAT3 inhibitor, normalized MDR1 gene expression in MMTV-PyMT;ApcMin/+ cells. Here, we hypothesize that loss of APC causes an increase in doxorubicin efflux and a decrease in DNA damage. First, we show that APC loss increased MDR1 activity as measured by calcein flux. To determine whether MDR1 inhibition would restore doxorubicin induced DNA damage, a combination treatment of doxorubicin and the MDR1 inhibitor, Valspodar, was able to restore doxorubicin sensitivity in MMTV-PyMT;ApcMin/+ cells compared to the control MMTV-PyMT;Apc+/+ cells. In addition, we have shown a decrease of γH2AX, a marker of damaged DNA, in MMTV-PyMT;ApcMin/+ cells treated with doxorubicin suggesting that the MMTV-PyMT;ApcMin/+ cells either have less damage due to increased drug efflux via MDR1 or due to increased DNA damage repair. Due to APC binding to Topoisomerase IIa, we also examined the degree of Topoisomerase IIa inhibition (via doxorubicin and etoposide) in MMTV-PyMT;ApcMin/+ compared to MMTV-PyMT;Apc+/+ cells. Topoisomerase IIa activity will also be measured to determine the effect of APC loss on Topoisomerase IIa. To test the hypothesis that loss of APC affected DNA damage repair, the expression of phosphorylated H2AX was measured in MMTV-PyMT;ApcMin/+ and MMTV-PyMT;Apc+/+ cells treated with the following: doxorubicin, etoposide, Valspodar, or a combination treatment. The longevity of doxorubicin-induced double stranded breaks was observed. Future studies will measure DNA damage repair pathway efficiency via reporter plasmids. We also will measure downstream signaling of yH2AX. Overall with an understanding of the role of APC in DNA damage repair, the use of a combination therapy could be advantageous to overcome chemotherapeutic resistance. Citation Format: Stephanie M. McClintock Batista. APC loss alters DNA damage repair in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 554.

Abstract 4463: Loss of APC mediates doxorubicin resistance in breast cancer cells

Abstract Resistance to chemotherapy is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for the advancement of novel therapeutic approaches to ultimately improve patient outcomes. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how loss of APC contributes to chemoresistance. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased cisplatin and doxorubicin-induced apoptosis. We previously showed that loss of APC in MMTV-PyMT;ApcMin/+ cells induced STAT3-activation resulting in an enhanced tumor initiating cell (TIC) population and increased multidrug resistance protein 1 (MDR1) expression. Therefore, we hypothesized that APC loss increased MDR1 activity and decreased doxorubicin-mediated DNA damage, ultimately preventing cell death. Using calcein incorporation, we first demonstrated that APC loss increased MDR1 activity which was normalized to WT control levels by the MDR1 inhibitor, Valspodar. We next showed that MDR1 inhibition in MMTV-PyMT;ApcMin/+ cells could restore doxorubicin-mediated apoptosis to that of MMTV-PyMT;Apc+/+ treated cells. Due to STAT3 inducing MDR1 activity and TIC population, MDR1 inhibition in MMTV-PyMT;ApcMin/+ cells was also shown to reduce the TIC population. In addition, we are investigating the effect of APC loss on DNA damage repair pathways. Preliminary studies have shown decreased γH2AX in MMTV-PyMT;ApcMin/+ cells treated with doxorubicin, suggesting a decrease in DNA damage. Downstream signaling of γH2AX, measured by phosphorylation of ATM, also demonstrated decreased DNA damage with ongoing studies measuring phosphorylation of ATR. Due to APC binding to topoisomerase IIα (topo IIα), a target of doxorubicin, the activity of topo IIα was measured to address whether this decreased DNA damage was attributed to impaired topo IIα activity or decreased intracellular doxorubicin due to increased MDR1 efflux. Future studies will investigate whether MDR1 inhibition in combination with doxorubicin will reduce tumor burden in vivo. Taken together, APC loss mediates doxorubicin resistance via enhanced MDR1 activity and altered DNA damage repair pathways demonstrating the potential use of combination therapy to overcome resistance to chemotherapy. Citation Format: Casey D. Stefanski, Kaitlyn Keffler, Stephanie McClintock, Jenifer R. Prosperi. Loss of APC mediates doxorubicin resistance in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4463.

Abstract 304: Dual PI3K/BCL-2 family inhibition in colorectal cancers with Apc and Pik3ca mutations

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer-related death in the United States. Approximately 20% of human CRCs possess mutations in the PIK3CA gene, resulting in a constitutively active form of phosphoinositide-3 kinase (PI3K). These PIK3CA mutations most commonly occur concomitantly with the loss of adenomatous polyposis coli (APC). Copanlisib is a beta-isoform sparing PI3K inhibitor. Here we examine the response of CRCs with Apc and Pik3ca mutations to copanlisib alone and in combination with navitoclax, a BCL-2, BCL-xL, and BCL-w inhibitor. Methods: Murine-derived organotypic cancer spheroids (MDOCS) with Apc and Pik3ca mutations generated from transgenic mice were cultured in Matrigel and treated by exchanging feeding media containing desired concentration of each agent over the spheroids. Spheroids were treated with feeding media (control), 200nM ABT263, 200nM copanlisib, or the combination of both treatments. The spheroid culture response was quantified as the median relative change in the sphere diameter, comparing pre- and posttreatment 4x optical microscopic images to those obtained 48 hours post-treatment. Results: Untreated MDOCS had a median growth in sphere diameter of 137%. ABT263 did not significantly affect growth; however, copanlisib significantly reduced median sphere size by 24.6% (p<0.001) and the combination treatment reduced median sphere size by 33% which was significant to both control and copanlisib only treated spheres (p<0.001 and p=0.014, respectively). Phosphorylation of ribosomal protein S6 and 4EBP1 were suppressed with copanlisib treatment. Induction of apoptosis in these spheres treated with the combination regimen was confirmed with immunofluorescence for cleaved caspase 3 with minimal apoptosis observed in those spheres treated with copanlisib alone. Conclusion: In this study, we demonstrated the ability of copanlisib alone and combined with ABT263 to cause a marked decrease in sphere size of CRC MDOCS with Apc and Pik3ca mutations. Future tests will examine this novel regimen in vivo and potentially in future clinical trials. Citation Format: Devon D. Miller, Christopher P. Babiarz, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Dual PI3K/BCL-2 family inhibition in colorectal cancers with Apc and Pik3ca mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 304.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wntsignaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Interaction of the Wnt/β-catenin and RAS-ERK pathways involving co-stabilization of both β-catenin and RAS plays important roles in the colorectal tumorigenesis.

Cancer development is usually driven by multiple genetic and molecular alterations rather than by a single defect. In the human colorectal cancer (CRC), series of mutations of genes are involved in the different stages of tumorigenesis. For example, adenomatous polyposis coli (APC) and KRAS mutations have been known to play roles in the initiation and progression of the tumorigenesis, respectively. However, many studies indicate that mutations of these two genes, which play roles in the Wnt/β-catenin and RAS-extra-cellular signal regulated kinase (ERK) pathways, respectively, cooperatively interact in the tumorigenesis in several different cancer types including CRC. Both Apc and Kras mutations critically increase number and growth rate of tumors although single mutation of these genes does not significantly enhance the small intestinal tumorigenesis of mice. Both APC and KRAS mutations even result in the liver metastasis with inductions of the cancer stem cells (CSCs) markers in a mice xenograft model. In this review, we are going to describe the history for interaction between the Wnt/β-catenin and RAS/ERK pathways especially related with CRC, and provide the mechanical basis for the cross-talk between the two pathways. The highlight of the crosstalk involving the stability regulation of RAS protein via the Wnt/β-catenin signaling which is directly related with the cellular proliferation and transformation will be discussed. Activation status of GSK3β, a key enzyme involving both β-catenin and RAS degradations, is regulated by the status of the Wnt/β-catenin signaling dependent upon extracellular stimuli or intracellular abnormalities of the signaling components. The levels of both β-catenin and RAS proteins are co-regulated by the Wnt/β-catenin signaling, and these proteins are overexpressed with a positive correlation in the tumor tissues of CRC patients. These results indicate that the elevation of both β-catenin and RAS proteins is pathologically significant in CRC. In this review, we also will discuss further involvement of the increments of both β-catenin and RAS especially mutant KRAS in the activation of CSCs and metastasis. Overall, the increments of β-catenin and RAS especially mutant KRAS by APC loss play important roles in the cooperative tumorigenesis of CRC.

Identification of Eendogenous <i>Adenomatous Polyposis Coli</i> Interaction Partners and &#946-Catenin-Independent Targets by Proteomics

Adenomatous polyposis coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of &#946-catenin, but the extent to which APC exerts Wnt/&#946-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and &#946-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of &#946-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 to APC mutant phenotypes. Collectively, our results highlight the extent and importance of Wntindependent APC functions in epithelial biology and disease.

Parafibromin, APC, and MIB-1 Are Useful Markers for Distinguishing Parathyroid Carcinomas From Adenomas.

Differentiation of parathyroid carcinoma (PC) from parathyroid adenoma (PA) relies solely on the pathologic determination of invasion of surrounding structures and/or distant metastasis. Parathyroid lesions with atypical histologic features with no demonstration of invasion or metastasis present a diagnostic dilemma. Different authors report a parafibromin and adenomatous polyposis coli (APC) loss or reduction in PC cases. High proliferative activity of MIB-1 and increased galectin 3 expression are reported in PC. There is no clear cutoff for the sensitivity, specificity, or predictive value for all these markers. The immunohistochemical expression of parafibromin, APC, MIB-1, and galectin 3 was studied in 73 adenomas, 21 PCs, and 3 atypical adenomas. The presence or absence of each marker was identified through the use of a comprehensive scoring system based on multiplying the percentage of tumor cells stained (0 to 100) and the staining intensity (0 to 3) on each biopsy. The highest score that any slide could reach was 300. A cutoff of >100 was used to consider the specimen positive for parafibromin, APC, or galectin 3 staining. MIB-1 proliferation indices were calculated using image cytometry; proliferation indices >5% were considered positive. We identified parafibromin loss in 7/21 (33%) carcinomas and 1/73 (1%) adenomas. Loss of APC was seen in 20/21 (95%) carcinomas and 38/73 (52%) adenomas. MIB-1 indices were elevated in 18/21 (86%) carcinomas. MIB-1 indices were <5% in all (100%) adenomas. MIB-1 indices were elevated in 2/3 (67%) atypical adenomas. Our study presents a clear cutoff to determine the practicality of using parafibromin, APC, and MIB-1 as immunohistochemical markers to differentiate between PCs and PAs. Loss of parafibromin and a high MIB-1 index are both independently sensitive and specific markers for the diagnosis of PC. Loss of APC was only specific for PC. This panel of markers provides a novel, useful approach in the diagnosis and differentiation of PCs from PAs.

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation.

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus – Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT;ApcMin/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT;ApcMin/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;ApcMin/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

Abstract A31: Targeting APC loss using synthetic lethality in Colorectal Cancer

Abstract Background: Mutations in the tumor suppressor gene Adenomatous Polyposis Coli (APC) are found in 80% of sporadic colorectal cancer (CRC) tumors and are also responsible for the inherited form of CRC, Familial Adenomatous Polyposis (FAP). APC mutations typically occur early in the development of CRC, followed by mutations in KRAS, SMAD2/3/4 and TP53. The majority of mutations in APC occur in a region known as the mutation cluster region (MCR). This region is responsible for negatively regulating levels of B-catenin and therefore the level of Wnt signaling activation. In APC mutated tumors, B-catenin is not tightly regulated resulting in hyperactivation of the Wnt signaling pathway leading to uncontrolled cellular proliferation, differentiation, migration and apoptosis. Aim: We aim to use the concept of synthetic lethality to identify novel therapeutic targets for the treatment of APC mutated CRC. Results: In order to identify novel therapeutic targets for the treatment of APC mutated CRC, we have generated an in vitro model of APC mutant CRC using a lentiviral CRISPR-cas9 system. Using the APC wildtype colorectal carcinoma cell line RKO, we targeted the cells with a guide RNA targeting the start of the final exon of APC, which covers 80% of the coding region. These cell lines allow us to identify targets, which are synthetically lethal with the APC mutation. To identify novel potential drug targets, we have used two parallel screening approaches. Firstly, we have screened our cell lines with a library of siRNA silencing over 700 kinases. Typically kinases are easy to target pharmaceutically, with many kinase inhibitors already clinically available. Upon analysis, we have identified seven genes which show synthetic lethality with the APC mutation, whilst not harming the control wildtype APC cells. In parallel, we have screened over 1000 FDA-approved compounds to identify drugs which cause increased selective lethality in APC mutant cells. From screening our FDA-approved compounds, we have identified a number of compounds which display synthetic lethality with the APC mutation. Conclusions: We have identified seven genes as potential therapeutic targets and a number of FDA-approved compounds, which could potentially be new selective therapies for 80% of CRC patients. Currently we are validating these findings and investigating the mechanism of synthetic lethality with APC mutation. To further validate our findings we are also exploring whether these results extend to other CRC cell lines with different mutational backgrounds, this will help us access how many patients may benefit from our novel therapeutic targets. Acknowledgments: We would like to thank Bowel and Cancer Research and The Rosetree Trust for co-funding this project. Citation Format: Hannah Shailes, Gemma Bridge, Daniel Foxler, Tyson V. Sharp, Andrew Silver, Sarah A. Martin. Targeting APC loss using synthetic lethality in Colorectal Cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A31.

Abstract 1081: APC regulation of breast cancer therapeutic resistance

Abstract Resistance to chemotherapy is one of the leading causes of death from breast cancer. Our lab discovered that Adenomatous Polyposis Coli (APC) loss in breast cancer cells results in an elevation of tumor-initiating cells (TICs) and resistance to chemotherapy-induced apoptosis. Given that TICs are often most resistant to standard chemotherapeutic compounds, we sought to understand the mechanism responsible for APC-mediated TIC enhancement. Our hypothesis is that the molecular mechanism involved in chemotherapeutic resistance parallels that promoting the TICs. APC-mutant cells have amplified activation of signal transducer and activator of transcription 3 (STAT3). Interestingly, inhibition of STAT3 with a small molecule inhibitor A69 decreases the TIC population and restores drug sensitivity. Studies are ongoing in the laboratory to investigate other molecular signaling pathways involved in APC-mediated enhanced TIC population and therapy resistance. These data have begun to reveal the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance. Note: This abstract was not presented at the meeting. Citation Format: Anne Arnason, Monica VanKlompenberg, Jenifer R. Prosperi. APC regulation of breast cancer therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1081. doi:10.1158/1538-7445.AM2017-1081

Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations.

Therapeutic targeting of the PI3K pathway is an active area of research in multiple cancer types, including breast and endometrial cancers. This pathway is commonly altered in cancer and plays an integral role in numerous vital cellular functions. Mutations in the PIK3CA gene, resulting in a constitutively active form of PI3K, often occur in colorectal cancer, though the population of patients who would benefit from targeting this pathway has yet to be identified. In human colorectal cancers, PIK3CA mutations most commonly occur concomitantly with loss of adenomatous polyposis coli (APC). Here, treatment strategies are investigated that target the PI3K pathway in colon cancers with mutations in APC and PIK3CA Colorectal cancer spheroids with Apc and Pik3ca mutations were generated and characterized confirming that these cultures represent the tumors from which they were derived. Pan and alpha isomer-specific PI3K inhibitors did not induce a significant treatment response, whereas the dual PI3K/mTOR inhibitors BEZ235 and LY3023414 induced a dramatic treatment response through decreased cellular proliferation and increased differentiation. The significant treatment responses were confirmed in mice with Apc and Pik3ca-mutant colon cancers as measured using endoscopy with a reduction in median lumen occlusion of 53% with BEZ235 and a 24% reduction with LY3023414 compared with an increase of 53% in controls (P < 0.001 and P = 0.03, respectively). This response was also confirmed with 18F-FDG microPET/CT imaging.Implications: Spheroid models and transgenic mice suggest that dual PI3K/mTOR inhibition is a potential treatment strategy for APC and PIK3CA-mutant colorectal cancers. Thus, further clinical studies of dual PI3K/mTOR inhibitors are warranted in colorectal cancers with these mutations. Mol Cancer Res; 15(3); 1-11. ©2016 AACR.

YAP–IL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis

Loss of tumor suppressor adenomatous polyposis coli (APC) activates β-catenin to initiate colorectal tumorigenesis. However, β-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.

Epithelial Membrane Protein 2 and β1 integrin signaling regulate APC-mediated processes

Adenomatous Polyposis Coli (APC) plays a critical role in cell motility, maintenance of apical-basal polarity, and epithelial morphogenesis. We previously demonstrated that APC loss in Madin Darby Canine Kidney (MDCK) cells increases cyst size and inverts polarity independent of Wnt signaling, and upregulates the tetraspan protein, Epithelial Membrane Protein 2 (EMP2). Herein, we show that APC loss increases β1 integrin expression and migration of MDCK cells. Through 3D in vitro model systems and 2D migration analysis, we have depicted the molecular mechanism(s) by which APC influences polarity and cell motility. EMP2 knockdown in APC shRNA cells revealed that APC regulates apical-basal polarity and cyst size through EMP2. Chemical inhibition of β1 integrin and its signaling components, FAK and Src, indicated that APC controls cyst size and migration, but not polarity, through β1 integrin and its downstream targets. Combined, the current studies have identified two distinct and novel mechanisms required for APC to regulate polarity, cyst size, and cell migration independent of Wnt signaling.

C-Cbl mediates the degradation of tumorigenic nuclear β-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors.

Despite the loss of Adenomatous Polyposis Coli (APC) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear β-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear β-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear β-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl's mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear β-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear β-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated β-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.