Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Chemotherapies against gastric cancer often fail, with cancer recurrence due potentially to the persistence of cancer stem cells. This unique subpopulation of cells in tumors possesses the ability to self-renew and dedifferentiate. These cancer stem cells are critical for initiation, maintenance, metastasis, and relapse of cancers; however, the molecular mechanisms supporting cancer stemness remain largely unknown. Increased kinase and decreased phosphatase activity are hallmarks of oncogenic signaling. Protein phosphatase 2A (PP2A) functions as a tumor-suppressor enzyme, and elevated levels of SET/I2PP2A, an endogenous PP2A protein inhibitor, are correlated with poor prognosis of several human cancers. Here, it was determined that SET expression was elevated in tumor tissue in a gastric cancer mouse model system, and SET expression was positively correlated with poor survival of human gastric cancer patients. Mechanistically, SET knockdown decreased E2F1 levels and suppressed the stemness of cancer cell lines. Immunoprecipitations show SET associated with the PP2A-B56 complex, and the B56 subunit interacted with the E2F1 transcription factor. Treatment of gastric cancer cells with the SET-targeting drug OP449 increased PP2A activity, decreased E2F1 protein levels, and suppressed stemness of cancer cells. These data indicate that a SET/PP2A/E2F1 axis regulates cancer cell stemness and is a potential target for gastric cancer therapy. This study highlights the oncogenic role of SET/I2PP2A in gastric cancer and suggests that SET maintains cancer cell stemness by suppressing PP2A activity and stabilizing E2F1. .

Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture from oncogene transgenic mouse gastrointestinal tissues was recently produced. However, ALI organoid culture system from tissues of colorectal cancer patients has not been established. Here, we developed an ALI organoid model from normal and tumor colorectal tissues of human patients. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Expression of an epithelial cell marker, E-cadherin, a goblet cell marker, MUC2, and a fibroblast marker, vimentin, but not a myofibroblast marker, α-smooth muscle actin (SMA), was observed in normal organoids. Expression of E-cadherin, MUC2, vimentin, and α-SMA was observed in tumor organoids. Expression of a cancer stem cell marker, LGR5 in tumor organoids, was higher than that in primary tumor tissues. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620, and HCT116. These findings indicate that ALI organoid culture from colorectal cancer patients may become a novel model that is useful for examining resistance to chemotherapy in tumor microenvironment.

ABSTRACT. Lymphoma is one of the most common malignant tumors in canine. Chemotherapy results in a high rate of remission; however, relapse and clinical drug resistance are usually seen within a year. Protein phosphatase 2A (PP2A) acts as a tumor suppressor and plays a critical role in mammalian cell transformation. Increased protein levels of SET, endogenous PP2A inhibitor, have been reported to correlate with poor prognosis in human leukemia. Here, we test the potential therapeutic role for a SET antagonist in canine lymphoma. We observed SET protein levels increased in multiple canine lymphoma cell lines compared with primary peripheral blood cells. A novel SET antagonist OP449 increased PP2A activity and effectively killed SET high-expressing canine lymphoma cells, but not SET low-expressing cells. Caspase-3 activation and enhanced Annexin V positive staining were observed after OP449 treatment, suggesting apoptotic cell death by OP449. Consistent with this, pan-caspase inhibitor Z-VAD-FMK blocked OP449 induced cell death. These data demonstrated the potential therapeutic application of SET antagonists for canine lymphoma. KEY WORDS: apoptosis, canine lymphoma, OP449, protein phosphatase 2A, SET.

We established 2 novel IMF cell lines from mouse colonic mucosa, namely, LmcMF and SmcMF, both of which were able to respond to LPS.

In colorectal cancer, gain-of-function mutations in KRas play a critical role in malignant transformation. Tumor growth in colorectal cancer is known to be promoted by the intestinal myofibroblasts (IMFs) that localize adjacent to the cancer cells, but the mechanisms of interaction between KRas-mutated cancer cells and the myofibroblasts remain unclear. Here, we investigated the effects of KRas-mutated cells on the behavior of myofibroblasts by using mouse primary IMFs and cells of an IMF cell line (LmcMF) and a mouse colon epithelial cell line (aMoC1). Conditioned medium (CM) was collected from aMoC1 cells overexpressing a control vector or KRasV12 vector (KRasV12-CM), and the effects of KRasV12-CM on IMFs were analyzed by performing proliferation assays, wound-healing assays, Boyden chamber assays, and western blotting. Whereas KRasV12-CM exerted little effect on the differentiation and proliferation of primary IMFs, the CM promoted migration of both primary IMFs and LmcMF cells. In KRasV12-overexpressing aMoC1 cells, mRNA expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was higher than in mock-transfected aMoC1 cells, and HB-EGF promoted the migration of primary IMFs and LmcMF cells. Moreover, KRasV12-CM-induced IMF migration was suppressed by dacomitinib, an inhibitor of HB-EGF receptors. Notably, in LmcMF cells, both KRasV12-CM and HB-EGF activated extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK), whereas KRasV12-CM-induced migration of IMFs was suppressed following treatment with either an ERK inhibitor (FR180204) or a JNK inhibitor (SP600125). These results suggest that HB-EGF secreted from KRas-mutated colorectal cancer cells promotes IMF migration through ERK and JNK activation, which, in turn, could support cancer progression.