SUMMARY Protein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis.
BackgroundMetastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver.ResultsLiver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 μg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 μg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 μg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid.ConclusionThese results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.
Sustained activation of PKC␣ is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKC␣ desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC 8 ) resulted in sustained plasma membrane association of PKC␣ in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKC␣, prolonged activation by DiC 8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC 8 -activated PKC␣ did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKC␣ desensitization. Although DiC 8 efficiently down-regulated novel PKCs PKC␦ and PKC⑀, differences in Ca 2؉ sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKC␣. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC 8 , targeted PKC␣ to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC 8 treatment failed to desensitize PKC␣ in several cell types and did not affect PKCI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKC␣ is seen in self-renewing epithelium of the intestine, cervix, and skin. PKC␣ deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKC␣ from desensitization in vivo.
Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer in women in the United States. Although loss of PTEN and deregulation of PI3K/AKT signaling has been implicated in approximately 90% of endometrial cancer cases, understanding of the molecular etiology of the disease remains limited. Our analysis of tissue from 448 endometrial cancer patients has determined that ∼60% of human tumors show reduced expression or loss of the signal transduction molecule PKCα. Loss of this enzyme is also seen in endometrial hyperplasias arising in mouse models carrying germline mutations of PTEN (PtenΔ4-5, PtenC124R and PtenG129E) or endometrial specific deletion of PTEN (Ptenpr-/−), indicating that loss of PKCα can be an early event in development of the disease. In patients, reduced expression of PKCα correlates with markers of disease aggressiveness, such as increased myometrial invasion and lymph node involvement, supporting a tumor suppressive role for the enzyme in the endometrium. This correlation is particularly marked in endometrioid, PTEN-mutant disease, pointing to a potential link between PKCα and PI3K/AKT signaling. The role of PKCα in endometrial cancer was further examined using a panel of 17 human endometrial cancer cell lines with varying expression of the kinase. Restoration of PKCα in cell lines lacking the enzyme blocked their ability to grow in soft agarose, further supporting a tumor suppressive role of the kinase in this tissue. Stimulation of cells that express PKCα with PKC agonists reduced phosphorylation/activation of AKT and promoted loss of downstream PI3K/AKT targets such as cyclin D1 and inhibitor of DNA binding 1 (Id1). Analysis of the PKC isozyme expression profile in these cells combined with the use of selective inhibitors pointed to PKCα as the mediator of the effect. The demonstration that PKC agonists failed to inhibit AKT signaling in cell lines that lack expression of PKCα, and that expression of exogenous PKCα restored the effect, confirmed the involvement of PKCα. The inhibition of AKT by PKCα is dependent on PP2A since okadaic acid and calyculin A, but not the PHLPP1/2 inhibitors NSC117079 and NSC45586, blocked the effect, a finding consistent with the ability of PKCα to inhibit AKT activity in PTEN mutant cell lines. Taken together, our study provides evidence that 1) loss of PKCα is a common and early event in endometrial cancer; 2) PKCα plays a crucial role in regulating AKT activation and growth promoting signaling molecules in endometrial cancer, and 3) effects of PKCα on AKT in endometrial cancer are mediated by PP2A. Given the importance of PI3K/AKT in the disease, these findings highlight the potential of PKCα signaling as a potential biomarker for disease risk and as a potential therapeutic target in endometrial cancer. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl Morrison, Baojing Chen, Subodh M. Lele, Takiko Daikoku, Sudhansu K. Dey, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) regulates PI3K/AKT signaling in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2015-4720
Endometrial cancer is the most common gynecological malignancy in the US and the fourth most common cancer in women. Despite its high incidence, there is limited knowledge of the molecular etiology of this disease. With obesity being a major risk factor, the incidence of endometrial cancer has been on a steady incline and is expected to become an increasing cause of cancer mortality in future years. Thus, improved understanding of endometrial tumorigenesis is of critical importance. Our analysis of human endometrial tumors identified loss of the signaling molecule PKCα in approximately 60% of endometrioid (Type I) tumors. Loss of PKCα trends with increasing tumor grade and is associated with aggressive disease features such as lymphovascular involvement and myometrial invasion, pointing to an important role for this molecule in regulating endometrial cancer progression. Inactivation of the tumor suppressor PTEN and deregulation of the PI3K/AKT pathway are key drivers of Type I endometrial cancer. Thus, mice with allele-specific knock-in of cancer-related Pten mutants (i.e., PtenΔ4-5, PtenC124R, and PtenG120E) offer unique models for the disease. Precancerous endometrial hyperplasias arising in these mice show loss of PTEN and increased AKT activity. While PKCα is expressed in the normal murine endometrial epithelium at all phases of the estrus cycle, the enzyme is uniformly lost in these precancerous lesions. Thus, disruption of PKCα signaling can occur early in endometrial tumorigenesis. In human endometrial cancer cell lines, low PKCα levels also correlate with PTEN loss; however, shRNA-mediated knockdown of PTEN in PTEN expressing cells did not affect PKCα expression indicating that downregulation of the kinase is not the direct result of changes in PTEN activity. Notably, PKC agonists suppress AKT activity in cells expressing high levels PKCα (HEC-1-A, HEC-50) but not in PKCα-low cells (Ishikawa, RL95-2). Collectively, these data suggest that, in addition to PTEN inactivation, loss of PKCα is required for full activation of the PI3K/AKT signaling pathway during endometrial carcinogenesis. Analysis of the molecular basis for loss of PKCα expression in endometrial tumors identified multiple regulatory mechanisms. PKCα protein expression in tumors generally parallels that of its mRNA and promoter activity assays point to transcriptional mechanisms for PKCα downregulation. Differential mRNA and protein stability were identified as additional levels of regulation of PKCα expression in endometrial cancer. Taken together, our findings indicate that 1) PKCα signaling regulates the PI3K/AKT pathway in the endometrial epithelium; 2) concomitant loss of PKCα and PTEN may act in a co-operative manner in regulation of endometrial turmorigenesis; and 3) loss of PKCα can occur by multiple mechanisms, as often seen with tumor suppressive molecules during neoplastic progression. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojing Chen, Subodh M. Lele, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) signaling in endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2014-4211
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