Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and in vivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

Stemness Is Enhanced in Gastric Cancer by a SET/PP2A/E2F1 Axis.

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.Implications: 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. Mol Cancer Res; 16(3); 554-63. ©2018 AACR.

The role of SET/I2PP2A in canine mammary tumors.

Canine mammary tumor is the most common neoplasm in female dogs, and it has generated considerable attention as a translational model for human breast cancer. Ser/Thr protein phosphatase 2A (PP2A) plays a critical role as a tumor suppressor, and SET/I2PP2A, the endogenous inhibitory protein of PP2A, binds directly to PP2A and suppresses its phosphatase activity. Here, we investigated the role of SET in the tumorigenic growth in canine mammary tumor as well as in the sensitivity of tumors to existing therapeutics. Elevated protein levels of SET were observed in advanced-stage of canine mammary tumor tissues of dogs compared with paired normal tissues. Knockdown of SET expression in a canine mammary tumor cell line CIP-m led to increased PP2A activity and decreased cell proliferation, colony formation, and in vivo tumor growth. We observed suppression of mTOR, ß-catenin, and NFκB signaling by SET knockdown. The sensitivity of CIP-m cells to doxorubicin was decreased by SET knockdown, while SET knockdown in CIP-m cells did not affect sensitivity to 4-OH-tamoxifen, carboplatin, bortezomib, and X-ray radiation. These data suggest that SET plays important roles in the tumor progression of a subset of canine mammary tumor by suppressing PP2A activity and enhancing mTOR, ß-catenin, and NFκB signaling.

Establishment of a Novel Model for Anticancer Drug Resistance in Three-Dimensional Primary Culture of Tumor Microenvironment.

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.

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma.

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.

Regulation of epithelial cell tight junctions by protease-activated receptor 2.

A layer of epithelial cells prevents the invasion of bacteria and the entry of foreign substances into the underlying tissue. The disruption of epithelial tight junctions initiates and exacerbates inflammation. However, the precise mechanism underlying the disruption of the epithelial tight junction remains unclear. The activation of protease-activated receptor 2 (PAR2) by serine proteases produced by some bacteria and mast cells contributes to inflammation in many tissues. In the present study, we tested the hypothesis that PAR2 activation affects the structure and function of tight junctions in Madin-Darby canine kidney (MDCK) cells. Although the application of a PAR2-activating peptide, PAR2-AP, from the apical side of MDCK cells failed to modify the transepithelial resistance (TER), its application from the basal side markedly suppressed the TER. In 3-dimensional cultures of MDCK cells expressing the mCherry-tagged PAR2, a lateral localization of PAR2 was observed. The application of PAR2-AP from the basal side changed the localization of the tight junctional protein, zonula occludin-1. Furthermore, PAR2-AP induced the phosphorylation of p38 MAP kinase. A p38 MAP kinase inhibitor, SB202190, inhibited PAR2-AP-induced changes in TER. Our results suggest that the activation of PAR2 leads to the disruption of tight junctions and increases the barrier permeability through the activation of p38 MAPK, which may cause the initiation and exacerbation of inflammation.