Protein kinase C epsilon promotes de novo lipogenesis and tumor growth in prostate cancer cells by regulating the phosphorylation and nuclear translocation of pyruvate kinase isoform M2.

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.

Inhibition of phosphoglycerate dehydrogenase induces ferroptosis and overcomes enzalutamide resistance in castration-resistant prostate cancer cells.

Phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in the first step of the serine synthesis pathway (SSP), is overexpressed in multiple types of cancers. The androgen receptor inhibitor enzalutamide (Enza) is the primary therapeutic drug for patients with castration-resistant prostate cancer (CRPC). However, most patients eventually develop resistance to Enza. The association of SSP with Enza resistance remains unclear. In this study, we found that high expression of PHGDH was associated with Enza resistance in CRPC cells. Moreover, increased expression of PHGDH led to ferroptosis resistance by maintaining redox homeostasis in Enza-resistant CRPC cells. Knockdown of PHGDH caused significant GSH reduction, induced lipid peroxides (LipROS) increase and significant cell death, resulting in inhibiting growth of Enza-resistant CRPC cells and sensitizing Enza-resistant CRPC cells to enzalutamide treatment both in vitro and in vivo. We also found that overexpression of PHGDH promoted cell growth and Enza resistance in CRPC cells. Furthermore, pharmacological inhibition of PHGDH by NCT-503 effectively inhibited cell growth, induced ferroptosis, and overcame enzalutamide resistance in Enza-resistant CRPC cells both in vitro and in vivo. Mechanically, NCT-503 triggered ferroptosis by decreasing GSH/GSSG levels and increasing LipROS production as well as suppressing SLC7A11 expression through activation of the p53 signaling pathway. Moreover, stimulating ferroptosis by ferroptosis inducers (FINs) or NCT-503 synergistically sensitized Enza-resistant CRPC cells to enzalutamide. The synergistic effects of NCT-503 and enzalutamide were verified in a xenograft nude mouse model. NCT-503 in combination with enzalutamide effectively restricted the growth of Enza-resistant CRPC xenografts in vivo. Overall, our study highlights the essential roles of increased PHGDH in mediating enzalutamide resistance in CRPC. Therefore, the combination of ferroptosis inducer and targeted inhibition of PHGDH could be a potential therapeutic strategy for overcoming enzalutamide resistance in CRPC.

ß2AR-HIF-1α-CXCL12 signaling of osteoblasts activated by isoproterenol promotes migration and invasion of prostate cancer cells.

BACKGROUND: Chronic stress is well known to promote tumor progression, however, little is known whether chronic stress-mediated regulation of osteoblasts contributes to the migration and invasion of metastatic cancer cells. METHODS: The proliferation, migration and invasion of prostate cancer cells were assessed by CCK-8 and transwell assay. HIF-1α expression of osteoblasts and epithelial-mesenchymal transition (EMT) markers of prostate cancer cells were examined by Western blot. The mRNA level of cytokines associated with bone metastasis in osteoblasts and EMT markers in PC-3 and DU145 cells were performed by qRT-PCR. Functional rescue experiment of cells were performed by using siRNA, plasmid transfection and inhibitor treatment. RESULTS: Isoproterenol (ISO), a pharmacological surrogate of sympathetic nerve activation induced by chronic stress, exhibited no direct effect on migration and invasion of PC-3 and DU145 prostate cancer cells. Whereas, osteoblasts pretreated with ISO promoted EMT, migration and invasion of PC-3 and DU145 cells, which could be inhibited by ß2AR inhibitor. Mechanistically, ISO increased the secretion of CXCL12 via the ß2AR-HIF-1α signaling in osteoblasts. Moreover, overexpression of HIF-1α osteoblasts promoted migration and invasion of PC-3 and DU145 cells, which was inhibited by addition of recombinant knockdown of CXCR4 in PC-3 and DU145 cells, and inhibiting CXCL12-CXCR4 signaling with LY2510924 blunted the effects of osteoblasts in response to ISO on EMT and migration as well as invasion of PC-3 and DU145 cells. CONCLUSIONS: These findings demonstrated that ß2AR-HIF-1α-CXCL12 signaling in osteoblasts facilitates migration and invasion as well as EMT of prostate cancer cells, and may play a potential role in affecting bone metastasis of prostate cancer.

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis.

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-ß-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.

Corrigendum: Pyruvate kinase M2 promotes prostate cancer metastasis through regulating ERK1/2-COX-2 signaling.

[This corrects the article DOI: 10.3389/fonc.2020.544288.].

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling.

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.

Pyruvate Kinase M2 Promotes Prostate Cancer Metastasis Through Regulating ERK1/2-COX-2 Signaling.

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis, which is highly expressed in many tumor cells, and has emerged as an important player in tumor progression and metastasis. However, the functional roles of PKM2 in tumor metastasis remain elusive. Here we showed that PKM2 promoted prostate cancer metastasis via extracellular-regulated protein kinase (ERK)-cyclooxygenase (COX-2) signaling. Based on public databases, we found that PKM2 expression was upregulated in prostate cancer and positively associated with tumor metastasis. Further analysis showed that PKM2 promoted prostate cancer cell migration/invasion and epithelial-mesenchymal transition (EMT) through upregulation of COX-2. Mechanistically, PKM2 interacted with ERK1/2 and regulated its phosphorylation, leading to phosphorylation of transcription factor c-Jun, downstream of ERK1/2, to activate COX-2 transcription by IP and ChIP assay, while inhibition of COX-2 significantly reversed the promotion effect of PKM2 on tumor metastasis in vivo. Taken together, our results suggest that a novel of PKM2-ERK1/2-c-Jun-COX-2 axis is a potential target in controlling prostate cancer metastasis.

A Conditional Knockout Mouse Model Reveals a Critical Role of PKD1 in Osteoblast Differentiation and Bone Development.

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.

Design, Synthesis, and Biological Evaluation of Substituted Pyrimidines as Potential Phosphatidylinositol 3-Kinase (PI3K) Inhibitors.

Three series of substituted pyrimidines were designed and synthesized. All target compounds were screened for kinase inhibitory activities against PI3Kα, and most IC50 values were found within the nanomolar range. Compounds 5d and 5p displayed comparable activities relative to the positive control 5a. 5p also showed a significant isozyme selectivity (PI3Kß/α). Furthermore, the cytotoxicities of these pyrimidines against human cancer cell lines were evaluated and the in vivo anticancer effect of 5d was also tested.

A novel small molecule aurora kinase inhibitor attenuates breast tumor-initiating cells and overcomes drug resistance.

Chemoresistance is a major cause of cancer treatment failure. Tumor-initiating cells (TIC) have attracted a considerable amount of attention due to their role in chemoresistance and tumor recurrence. Here, we evaluated the small molecule Aurora kinase inhibitor AKI603 as a novel agent against TICs in breast cancer. AKI603 significantly inhibited Aurora-A (AurA) kinase and induced cell-cycle arrest. In addition, the intragastric administration of AKI603 reduced xenograft tumor growth. Interestingly, we found that breast cancer cells that were resistant to epirubicin expressed a high level of activated AurA and also have a high CD24(Low)/CD44(High) TIC population. The inhibition of AurA kinase by AKI603 abolished the epirubicin-induced enrichment of TICs. Moreover, AKI603 suppressed the capacity of cells to form mammosphere and also suppressed the expression of self-renewal genes (ß-catenin, c-Myc, Sox2, and Oct4). Thus, our work suggests the potential clinical use of the small molecule Aurora kinase inhibitor AKI603 to overcome drug resistance induced by conventional chemotherapeutics in breast cancer.