Ron knockdown and Ron monoclonal antibody IMC-RON8 sensitize pancreatic cancer to histone deacetylase inhibitors (HDACi).

Recepteur d\u27origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer

Intra-tumoral heterogeneity in metastatic potential and survival signaling between iso-clonal HCT116 and HCT116b human colon carcinoma cell lines.

BACKGROUND: Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS). MATERIALS AND METHODS: HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days. RESULTS: Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death. CONCLUSIONS: We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer

TGFβ/Smad3 regulates proliferation and apoptosis through IRS-1 inhibition in colon cancer cells.

In this study, we have uncovered a novel crosstalk between TGFβ and IGF-1R signaling pathways. We show for the first time that expression and activation of IRS-1, an IGF-1R adaptor protein, is decreased by TGFβ/Smad3 signaling. Loss or attenuation of TGFβ activation leads to elevated expression and phosphorylation of IRS-1 in colon cancer cells, resulting in enhanced cell proliferation, decreased apoptosis and increased tumor growth in vitro and in vivo. Downregulation of IRS-1 expression reversed Smad3 knockdown-mediated oncogenic phenotypes, indicating that TGFβ/Smad3 signaling inhibits cell proliferation and increases apoptosis at least partially through the inhibition of IRS-1 expression and activation. Additionally, the TGFβ/Smad3/IRS-1 signaling axis regulates expression of cyclin D1 and XIAP, which may contribute to TGFβ/Smad3/IRS-1-mediated cell cycle progression and survival. Given that loss of TGFβ signaling occurs frequently in colon cancer, an important implication of our study is that IRS-1 could be a potential therapeutic target for colon cancer treatment

Transforming growth factor-β suppresses metastasis in a subset of human colon carcinoma cells.

BACKGROUND: TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling. METHODS: To test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging. RESULTS: Abrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma. CONCLUSIONS: The observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity

Colon carcinoma cells harboring PIK3CA mutations display resistance to growth factor deprivation induced apoptosis.

PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival

Active Learning Pipeline for Brain Mapping in a High Performance Computing Environment

This paper describes a scalable active learning pipeline prototype for large-scale brain mapping that leverages high performance computing power. It enables high-throughput evaluation of algorithm results, which, after human review, are used for iterative machine learning model training. Image processing and machine learning are performed in a batch layer. Benchmark testing of image processing using pMATLAB shows that a 100$\times$ increase in throughput (10,000%) can be achieved while total processing time only increases by 9% on Xeon-G6 CPUs and by 22% on Xeon-E5 CPUs, indicating robust scalability. The images and algorithm results are provided through a serving layer to a browser-based user interface for interactive review. This pipeline has the potential to greatly reduce the manual annotation burden and improve the overall performance of machine learning-based brain mapping.Comment: 6 pages, 5 figures, submitted to IEEE HPEC 2020 proceeding

Characterization of CDK(5) Inhibitor, 20-223 (aka CP668863) for Colorectal Cancer Therapy

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 – now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was ~3.5-fold and ~65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average \u3e2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy

Identification of a Novel TGFβ/PKA Signaling Transduceome in Mediating Control of Cell Survival and Metastasis in Colon Cancer

Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors

Transforming growth factor-β suppresses metastasis in a subset of human colon carcinoma cells

BACKGROUND: TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling. METHODS: To test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging. RESULTS: Abrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma. CONCLUSIONS: The observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity