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KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS (G12V) expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.

Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.

Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease. [ABSTRACT FROM AUTHOR]