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Protein kinase C epsilon (PKCε) belongs to a family of serine/threonine kinases that control cell proliferation, differentiation and survival. Aberrant PKCε activation and overexpression is a frequent feature of numerous cancers. However, its role in regulation of lipid metabolism in cancer cells remains elusive. Here we report a novel function of PKCε in regulating of prostate cancer cell proliferation by modulation of PKM2-mediated de novo lipogenesis. We show that PKCε promotes de novo lipogenesis and tumor cell proliferation via upregulation of lipogenic enzymes and lipid contents in prostate cancer cells. Mechanistically, PKCε interacts with NABD (1-388) domain of C-terminal deletion on pyruvate kinase isoform M2 (PKM2) and enhances the Tyr105 phosphorylation of PKM2, leading to its nuclear localization. Moreover, forced expression of mutant Tyr105 (Y105F) or PKM2 inhibition suppressed de novo lipogenesis and cell proliferation induced by overexpression of PKCε in prostate cancer cells. In a murine tumor model, inhibitor of PKM2 antagonizes lipogenic enzymes expression and prostate cancer growth induced by overexpression of PKCε in vivo. These data indicate that PKCε is a critical regulator of de novo lipogenesis, which may represent a potential therapeutic target for the treatment of prostate cancer.

BackgroundDermal fibrosis occurs in many human diseases, particularly systemic sclerosis (SSc) where persistent inflammation leads to collagen deposition and fiber formation in skin and multiple organs. The family of protein kinase D (PKD) has been linked to inflammatory responses in various pathological conditions, however, its role in inflammation-induced dermal fibrosis has not been well defined. Here, using a murine fibrosis model that gives rise to dermal lesions similar to those in SSc, we investigated the role of PKD in dermal fibrosis in mice lacking PKD2 activity.MethodsHomozygous kinase-dead PKD2 knock-in mice (PKD2SSAA/SSAA-KI) were obtained through intercrossing mice heterozygous for PKD2S707A/S711A(PKD2SSAA). The wild-type and KI mice were subjected to repeated subcutaneous injection of bleomycin (BLM) to induce dermal inflammation and fibrosis. As controls, mice were injected with PBS. At the end of the experiment, mouse skin at the injection site was dissected, stained, and analyzed for morphological changes and expression of inflammatory and fibrotic markers. PKD-regulated signaling pathways were examined by real-time RT-qPCR and Western blotting. In a separate experiment, BLM-treated mice were administered with or without a PKD inhibitor, CRT0066101 (CRT). The effects of CRT on dermal fibrosis were analyzed similarly. The identity of the PKD expressing cells were probed using myeloid lineage markers CD45, CD68 in BLM-treated mouse tissues.ResultsDermal thickness and collagen fibers of kinase-dead PKD2-KI mice were significantly reduced in response to BLM treatment as compared to the wild-type mice. These mice also exhibited reduced α-smooth muscle actin (α-SMA) and collagen expression. At molecular levels, both transforming growth factor β1 (TGF-β1) and interleukin-6 (IL-6) mRNAs were decreased in the KI mice treated with BLM as compared to those in the wild-type mice. Similarly, CRT significantly blocked BLM-induced dermal fibrosis and inhibited the expression of α-SMA, collagen, and IL-6 expression. Further analysis indicated that PKD2 was mainly expressed in CD45+/CD68+myeloid cells that could be recruited to the lesional sites to promote the fibrotic process of the skin in response to BLM.ConclusionsKnock-in of the kinase-dead PKD2 or inhibition of PKD activity in mice protected against BLM-induced dermal fibrosis by reducing dermis thickness and expression of fibrotic biomarkers including α-SMA, collagen, and inflammatory/fibrotic mediators including TGF-β1 and IL-6. PKD2 does this potentially through modulating the recruitment and function of myeloid cells in skin of BLM-treated mice. Overall, our study demonstrated a potential critical role of PKD catalytic activity in inflammation-induced dermal fibrosis.

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.

The protein kinase D family of serine/threonine kinases, particularly PKD1, has been implicated in the regulation of a complex array of fundamental biological processes. However, its function and mechanism underlying PKD1-mediated the bone development and osteoblast differentiation are not fully understood. Here we demonstrate that loss of PKD1 function led to impaired bone development and osteoblast differentiation through STAT3 and p38 MAPK signaling using in vitro and in vivo bone-specific conditional PKD1-knockout (PKD1-KO) mice models. These mice developed markedly craniofacial dysplasia, scapula dysplasia, long bone length shortage and body weight decrease compared with wild-type littermates. Moreover, deletion of PKD1 in vivo reduced trabecular development and activity of osteoblast development, confirmed by Micro-CT and histological staining as well as expression of osteoblastic marker (OPN, Runx2 and OSX). Mechanistically, loss of PKD1 mediated the downregulation of osteoblast markers and impaired osteoblast differentiation through STAT3 and p38 MAPK signaling pathways. Taken together, these results demonstrated that PKD1 contributes to the osteoblast differentiation and bone development via elevation of osteoblast markers through activation of STAT3 and p38 MAPK signaling pathways.