Overexpression, clinical significance and potential mechanisms of protein kinase D1 in hepatocellular carcinoma: multi-omic analyses and pharmacological insights.

OBJECTIVE: Protein Kinase D1 (PKD1) plays an important role in several cellular processes such as cell proliferation, oxidative stress signaling, adhesion and motility. PKD1 is of particular interest in diagnosis, prognosis and treatment of human diseases because of its enzymatic activity and susceptibility to successful therapeutic targeting. In our earlier study we have demonstrated suppression of PKD1 expression in advance stage prostrate cancer. This led us to analyze PKD1 expression patterns in various other cancer tissue samples. MATERIAL AND METHODS: Tissue microarrays containing various cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry (Biocare Medical, Concord, CA). Further studies on PKD1 expression profile in hepatocellular carcinoma (HCC) were carried out using tissue microarray (TMA) containing HCC samples (n=70) with normal tissues (n=4) and a HCC TMA with hepatitis B viral history (HBV) containing HCC samples (n=100) with normal tissues (n=4) (AccuMax Array). Immunostaining was graded by two independent pathologists. Intensity and extent of staining scores were multiplied to obtain the composite score (CS) of each sample. RESULT: Differential expression pattern of PKD1 was observed in brain, skin, lung, kidney, thyroid and ovarian cancer samples. Along with stomach and colon cancer, HCC samples showed a marked downregulation of PKD1 expression compared to their respective normal tissues. PKD1 expression profile was further evaluated in a large cohort of HCC samples. Overall, HCC samples showed a significant (p>0.05) downregulation (Mean CS=6.0) of PKD1 expression compared to normal liver samples (Mean CS=11.4). Among cancer samples, advanced stage samples showed a relatively lower PKD1 expression (Mean CS=5.4) compared to early stage (Mean CS=6.6) HCC samples. Similarly, advance stage HCC samples with HBV showed a lower PKD1 expression (Mean CS=6.9) compared to early stage samples (Mean CS=9.6) and non neoplastic samples (Mean CS=12). CONCLUSION: The results of this study suggest suppression of PKD1 expression in HCC. In view of the limitations of the available diagnostic/prognostic studies for detection of HCC, the aberrant expression of PKD1 may serve as a novel biomarker for the diagnosis/prognosis of hepatocellular carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4621.

Introduction: Prostate cancer (PCa), one of the most common cancers among men, initially manifests as an androgen-dependent malignancy. PCa eventually and inevitably progresses into hormone refractory or castration-resistant condition. Relapse of castration-resistant prostate cancer (CRPC) is life threatening, and responsible for the majority of mortality among PCa patients. During the transition from the hormone naive disease into CRPC, there is an up regulation of cell cycle markers and a decline in the expression of tumor suppressors like protein kinase D1 (PrKD1). The role of PrKD1 as a tumor suppressor has been identified in regulating various cellular events including the inhibition of the mitogenic pathways induced by growth factors and androgens. However, the potential role of PrKD1 as a cell cycle regulator has not been explored in solid cancers including prostate. Therefore, the present study has been designed to investigate the role of PrKD1 as a cell cycle regulator in PCa cells. Methods: LNCaP cells with high expression of PrKD1 and its aggressive derivative C4-2 cells with low expression of PrKD1 were used in this study. To evaluate the direct effect of PrKD1, C4-2 cells were stably transfected with PrKD1 and used alongside the two other cell lines. Cells were cultured in the presence and absence of Bryostatin 1 (Bryo-1, a PrKD1 activator) for 30 hours and exposed to 10 Gray gamma radiation (IR) to induce DNA damage. After the treatment period, cell cycle was assessed using flow cytometry and the phosphorylation of Cdc25 phophatases were analyzed by western blot. All experiments were repeated after inhibiting check point kinase 1 and 2 (CHEK1 and CHEK2) with siRNA to evaluate the independency of PrKD1 action on cell cycle. Results: LNCaP cells underwent cell cycle arrest in both Bryo-1 and IR group, whereas non-transfected C4-2 cells responded only to IR, not to Bryo-1. However, C4-2 cells when transfected with PrKD1 were arrested at G1/S checkpoint in response to Bryo-1. Down regulation of CHEK1 and CHEK2 with SiRNA in C4-2 cells did not cause any significant cell cycle arrest in response to DNA damage due to IR. Over expression of PrKD1 in these cells rescued the effect of siRNA against CHEK1 and CHEK2. Western blot analysis showed that activation of PrKD1 with Bryo-1 causes phophorylation of Cdc25 C phophatase. Conclusion: Our study showed that PrKD1 phosphorylates CDC25C and induces cell cycle arrest in G1/S phase in response to DNA damage. The results suggest that PrKD1, like the two other Serine Threonine kinases (CHEK1 and CHEK2), mediates cell cycle arrest in response to DNA damage. Citation Format: Bita Nickkholgh, Sivanandane Sittadjody, Michael B. Rothberg, KC Balaji. Protein kinase D1 induces cell cycle arrest independent from check point kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-243.

Protein kinase D1 (PRKD1) is down regulated in gastric, breast and prostate cancer. Several regulatory mechanisms modulate PRKD1 activity in cancers. In colon cancer, it has been shown that PRKD1 is down regulated by nuclear beta-catenin. We explored whether beta-catenin is involved in regulation of PRKD1 expression in prostate cancer as well. A CHIP assay performed using prostate cancer LNCaP cell lysate and pull down with beta -catenin antibody, which demonstrated recruitment of beta -catenin to promoter of PRKD1 gene as well as at downstream regions in the gene suggesting that beta -catenin could be involved in PRKD1 regulation. To identify the exact binding site of beta-catenin to PRKD1 gene, we carried out CHIP sequencing. The beta -catenin protein complex was bound to a 166 bp sequence near exon 2 (chr14:29899631-29899796). Because beta -catenin is a coactivator and not a known transcription factor, we performed a transcription factor promoter array, which showed that the MYC/MAX transcription factor complex may be the mediator of the regulatory effect of beta -catenin on PRKD1 expression. We validated the results by performing CHIP assay using MYC and MAX antibodies which showed recruitment of both MYC and MAX to the same beta -catenin binding site to PRKD1. In order to assess the functional impact of beta -catenin regulation of PRKD1 in prostate cancer, beta-catenin mutated at threonine 120 residue (we have previously shown that PRKD1 is the only known kinase to phosphorylate the site) demonstrated increased nuclear translocation of beta-catenin and increase down regulation of PRKD1 and increased androgen receptor (AR) activity (increased AR activity is well established to be associated with PC progression). Our data has identified a novel auto-regulatory mechanism of PRKD1 expression through beta-catenin phosphorylation. Citation Format: Bita Nickkholgh, Sittadjody Sivanandane. Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2017-5498

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Objective: Pancreatic cancer is one of the deadliest diseases, and the fourth most common cause of cancer-related deaths in the United States. Protein Kinase D1 (PKD1), a serine-threonine kinase, is an important modulator of several kinase signal transduction pathways. Although, PKD1 is known to be involved in pancreatic cancer pathogenesis, the underlying signaling mechanisms are largely unknown. Therefore, we investigated how PKD1 contributes to pancreatic tumor growth and progression. Our studies suggest a novel role of PKD1 in regulating glucose metabolism in pancreatic cancer, which drives pancreatic tumorigenesis and progression. Methods: Glucose and Lactate assays were performed in pancreatic cancer cells following PKD1 transfection. Cell culture media was collected after 48 hrs to measure the amount of glucose consumption and L-lactate production on PKD1 expression in pancreatic cancer cells using in vitro assay kits (Cayman Chemicals). Immunoblotting and qRT-PCR assays were performed to assess the expression of protein and mRNA levels, respectively, of key signaling molecules in glucose metabolism in pancreatic cancer cells. Cell proliferation and colony forming assays were performed to determine the effect of PKD1 on cell proliferation and survival under both normoxic and hypoxic conditions. In vitro functional assays for investigating migration and invasion were performed using boyden chamber and Matrigel assays, respectively, under normoxic and hypoxic conditions. Results: Our results show that PKD1 leads to altered glucose metabolism in pancreatic cancer cells. We observed increased amount of L-lactate production and glucose consumption on PKD1 expression in cells. This indicates the alterations in glucose metabolism associated with PKD1 expression in cells. These alterations were accompanied with enhanced expression of both protein and mRNA levels of Glut-1, HIF-1α, and KRAS, which are associated with glucose metabolism. These events were observed in both normoxic and hypoxic conditions, indicating that the PKD1 modulated events are independent of oxygen tension. Furthermore, we observed enhanced invasion and migration of pancreatic cancer cells on PKD1 expression, which was further increased by addition of lactate (an end product of aerobic glycolysis). Altogether, our studies indicate a role of PKD1 as a key regulator of the glucose metabolism and promoter of pancreatic cancer oncogenesis. Conclusion: These results suggest that PKD1 plays an important role in metabolic reprogramming of pancreatic cancer cell metabolism to induce cancer growth and enhanced cellular invasion and motility. This is a first study that suggests the involvement of PKD1 in metabolic remodeling of pancreatic cancer cells leading to enhanced tumor growth and progression. Citation Format: Sonam Kumari, Sheema Khan, Murali M. Yallapu, Subhash C. Chauhan, Meena Jaggi. Aberrant expression of protein kinase D1 influences metabolic reconditioning in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1449.

Pancreatic oncogenic signaling cascades converge at Protein Kinase D1

Background: Prostate cancer (PrCa) is the second leading cause of cancer-related deaths in American Men. Docetaxel (DTX) is a standard first-line treatment for metastatic castration-resistant PrCa after the failure of hormone therapy. However, most PrCa patients who receive DTX experience only transient benefits and rapidly develop incurable drug resistance. Protein Kinase D1 (PKD1), one of the serine threonine kinases from PKD family is highly expressed in normal prostate tissues and is suppressed during PrCa progression. Accumulative evidence suggest a tumor suppressive role of PKD1 in PrCa, while other isoforms of PKD (PKD2 and PKD3) act as oncogene. In this study, we identified pharmacological agent Ormeloxifene (ORM) which selectively activates PKD1 and inhibits metastasis associated protein 1 (MTA1), thus induces sensitivity to DTX treatment in PrCa cells. Materials and Methods: We have used androgen-independent human PrCa cells (C4-2) which show low PKD1 expression compared to other PrCa cell lines. Cells were treated with 10 and 15 μM doses of ORM for 24 hrs and various functional assays (cell proliferation, colony formation, motility and invasion) were performed. In a parallel experiment, cells were treated with ORM (10 and 15 μM) for 24 hrs protein and RNA samples isolation. Protein lysates were used to investigate the effect of ORM on PKD1, PKD2, PKD3 and MTA1 protein levels. qRT-PCR was performed to investigate the effect of ORM on PKD1, PKD2 and PKD3 expression at mRNA levels. To investigate if ORM treatment sensitizes the effects of DTX, cells were treated with ORM and DTX alone or in combination. In-silico docking studies were performed to determine the putative molecular interaction of ORM with MTA1. Results: ORM treatment inhibits proliferation and clonogenic potential of C4-2 cells. We observed that ORM significantly induces PKD1 expression at protein and mRNA level in C4-2 cells. To determine whether this PKD1 inducing effects of ORM in PrCa cells is specific, we examined the effects of ORM on PKD2 and PKD3 at mRNA and protein levels. Interestingly, we observed that ORM treatment inhibits expression of oncogenic PKD3 isoform, however, no effect PKD2 was observed. MTA1 is involved in DTX resistance and ORM treatment effectively inhibited the expression of MTA1. However, there was no effect of DTX treatment on the expression of MTA1. We also observed that ORM treatment significantly potentiates the effect of DTX on cell viability and colony formation of C4-2 cells. In-silico docking studies between ORM and MTA1 showed four potential binding sites with best score at serine 270. Conclusion: Overall, our study defines ORM as a novel PKD1 activator/modulator which also inhibits a key metastasis associated protein, MTA1 and sensitizes the PrCa cells to DTX. Based on these results, it appears that ORM may be a novel therapeutic modality for advanced stage metastatic PrCa alone or in combination with DTX. Citation Format: Aditya Ganju, Bilal Bin Hafeez, Fathi Halaweish, Wei Li, Man Mohan Singh, Murali Mohan Yallapu, Subhash Chauhan, Meena Jaggi. Ormeloxifene, a novel pharmacological activator of PKD1 enhances docetaxel sensitivity in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3862.

Objective:This study aimed to assess protein kinase D1 expression and its association with tumor characteristics as well as prognosis in patients with non-small cell lung cancer.Methods:Protein kinase D1 expression in tumor tissues and adjacent tissues from 172 patients with non-small cell lung cancer who underwent surgical resection were analyzed by immunohistochemical staining. Based on the total immunohistochemical score, protein kinase D1 expression was classified as protein kinase D1 high expression (further divided into protein kinase D1 high+++, protein kinase D1 high++, and protein kinase D1 high+ expressions) and protein kinase D1 low expression. Clinical characteristics of patients with non-small cell lung cancer were acquired from the database. Accumulating disease-free survival and overall survival were calculated based on patients’ relapse/survival status.Results:Protein kinase D1 expression was increased in tumor tissues compared to adjacent tissues (P < .001). Tumor protein kinase D1 high expression correlated with poorer pathological differentiation (P = .041), increased tumor size (P = .003), the presence of lymph node metastasis (P = .001), and elevated tumor, nodes and metastases stage (P < .001). Besides, both accumulating disease-free survival and overall survival were decreased in patients with tumor protein kinase D1 high expression compared to patients with tumor protein kinase D1 low expression (P = .010 for disease-free survival and P = 0.005 for overall survival). Moreover, they were lowest in patients with tumor protein kinase D1 high+++ expression, followed by patients with tumor protein kinase D1 high++ expression, then patients with tumor protein kinase D1 high+ expression, and highest in patients with tumor protein kinase D1 low expression (P < .001 for disease-free survival and P = .001 for overall survival). Notably, higher tumor protein kinase D1 expression was an independent predictive factor for decreased disease-free survival (P = .001) and overall survival (P = .004).Conclusions:Protein kinase D1 might be a potential marker to identify patients with non-small cell lung cancer with worse tumor features and prognosis.

Objective: Protein Kinase D1 (PKD1) is an important modulator of several signal-transduction pathways in benign and malignant human diseases. Currently the role of PKD1 in colon cancer, which is the second-leading cause of cancer-related deaths, is not well established. We have found previously that advance stage prostate cancer has suppressed expression. This led us to analyze PKD1 expression patterns in various other cancer tissue samples including colon cancer. Materials and Methods: Tissue microarrays containing colon cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry. To determine PKD1 mediated effects on oncogenic β-catenin signaling in colon cancer, human colorectal adenocarcinoma cell line SW480. Stable transformants of SW480 were created with GFP and SW480-PKD1-GFP, PKD1 overexpression leads to attenuation of β-catenin/T cell factor (TCF) transcription activity measured by luciferase reporter assays. To determine the effect of PKD1 overexpression on other cancer associated genes, RT-PCR array (that had 84 cancer related genes) analysis was performed in SW480-GFP and SW480-PKD1-GFP cells. Results: Our expression analysis determined a significant decrease in PKD1 expression in advanced Dukes stage (II, III and IV) colon cancer samples as compared to non-neoplastic colon samples. We have also noticed downregulation and aberrant localization of β-catenin in colon cancer samples compared to non-neoplastic colon samples. Therefore, we also determined the function of PKD1 in oncogenic β-catenin signaling in colon cancer. Our confocal microscopy analyses demonstrates that PKD1 up-regulation caused translocation of β-catenin from the nucleus to the plasma membrane. PKD1 overexpression also reduced β-catenin/TCF interaction in the nucleus and suppressed the expression of its downstream signaling proteins. Phenotypic changes in the stable transformants were measured via proliferation assays, anchorage independent growth, anchorage dependent growth, cellular motility and invasion. The overexpression of PKD1 markedly suppressed cell proliferation in SW480 cells. The SW480-PKD1-GFP cells formed fewer colonies compared with SW480-GFP cells. The SW480-PKD-GFP cells showed very minimal or no motility and invasion compared to SW480-GFP cells. Our RT-PCR array data shows that PKD1 overexpression also modulates expression of tissue inhibitor of metalloproteinase 3 (TIMP3), interferon α, interferon β and tumor necrosis factor receptor (TNFR). Conclusion: In conclusion, PKD1 is down regulated in colon cancer and its downregulation is correlated with aberrant β-catenin subcellular localization. Our data suggest that the altered PKD1 expression may induce β-catenin dependent and independent signaling in colon cancer and suppression of PKD1 may play a critical role in colon carcinogenesis and colon cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2907. doi:10.1158/1538-7445.AM2011-2907

PKD1 is downregulated in non-small cell lung cancer and mediates the feedback inhibition of mTORC1-S6K1 axis in response to phorbol ester

Objective: Pancreatic cancer (PanCa) is the third most common cause of cancer-related deaths in the US. Protein Kinase D1 (PKD1) is a kinase molecule which is involved in various important cellular signaling processes. While PKD1 has been reported to play a role in PanCa progression, the molecular mechanisms involved have not been adequately studied. Herein, we investigate the underlying mechanisms which enable PKD1 in enhancing glucose metabolism and further contribute to PanCa aggressiveness. Methods: PanCa cells with high PKD1 expressing (Panc-1 and AsPC-1) and low PKD1 (HPAF-II and BXPC-3) were used in the study. Immunohistochemistry and confocal immunofluorescence were performed to analyze the expression of PKD1 in pancreatic cancer/normal tissues and cells, respectively. The effect of PKD1 gain/loss-in function was investigated on glucose uptake and lactate production in PanCa cells using commercially available kits. Western blotting and real-time PCR experiments were performed to analyze the expression of protein and mRNA levels. MTT assay was conducted to access the influence of PKD1 on cell proliferation. Boyden chamber migration assay and Matrigel invasion assays were performed to determine the migratory and invasive abilities of PanCa cells. The gene silencing was performed using specific siRNAs in the study. Results: Our results demonstrate that PKD1 upregulates glucose metabolism in PanCa cells as indicated by enhanced glucose consumption and lactate production. The invasive characteristics of PKD1 expressing cells are augmented in presence of L-Lactate and reduced in presence of 2DG. PKD1 overexpression demonstrated enhanced phosphorylation of mTOR (ser-2448), 4EBP1, s6kinase and AKT, suggesting the role of PKD1 in the activation of mTOR pathway. We further observed that the phosphorylation of mTOR (ser-2448) and ps6kinase was attenuated on silencing PKD1 or in presence of Rapamycin, suggesting the role of PKD1 in activation of mTORC1 complex, both being the main effectors of mTORC1. Additionally, the inhibition in the phosphorylation of mTOR (ser-2448) in presence of kinase dead PKD1 suggests that PKD1 phosphorylates/activates mTORC1 in PanCa. Decrease in glucose uptake and lactate production in presence of PKD1 overexpression was observed on silencing raptor but not rictor in the cells, further suggesting the involvement of mTORC1 in PKD1 induced metabolic reprogramming in PanCa. PKD1 induced enhanced phosphorylation of mTOR resulted in an activation of HIF-1α and Glut-1 proteins, involved in abberant glucose metabolism. Additionally, our results also demonstrate that altered glucose metabolism leads to chemoresistance and silencing of PKD1 expression sensitizes PanCa cells to gemcitabine. Conclusion: Our studies indicate that PKD1 acts as a key regulator of the glucose metabolism via mTOR activation and facilitates proliferation and invasion of PanCa cells. Citation Format: Sonam Kumari, Sheema Khan, Murali Yallapu, Subhash Chauhan, Meena Jaggi. Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4367.

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of protein kinase D1 (PKD1) in a cutaneous squamous cell carcinoma cell line A431, and to explore the mechanism underlying ALA-PDT-induced apoptosis of A431 cells. Methods A431 cells were cultured in vitro, and cell counting kit-8 (CCK-8) assay was performed to select the optimal combination of ALA concentration and PDT dose with the strongest proliferation inhibitory effect. A431 cells at exponential growth phase were randomly divided into 4 groups: control group receiving no treatment, ALA group treated with ALA solution alone, PDT group treated with PDT alone, and ALA-PDT group treated firstly with ALA solution and then with PDT. After 12-, 24-, 36- and 48-hour additional culture, CCK-8 assay was conducted to evaluate the cellular proliferation inhibition, and the apoptosis rate at the time point of the strongest proliferation inhibitory effect was measured by flow cytometry. RT-PCR was performed to determine the expression of protein kinase D1 gene (PRKD1) in A431 cells at different time points after the ALA-PDT treatment, and Western blot analysis to measure protein expression of PKD1 and its phosphorylation at Tyr463 (pTyr463) and Ser916 (pSer916) in A431 cells. Results The combi-nation of ALA at the concentration of 1.5 mmol/L with PDT at an irradiation dose of 2 J/cm2 was optimal due to its strongest proliferation inhibitory effect. After 12-, 24-, 36- and 48-hour additional culture, there were significant differences in the proliferation inhibition rate among the 4 groups (F= 39.56, P 0.05) . No significant difference in the Ser916-phosphorylated PKD1 expression was found among the 4 groups (F= 1.53, P > 0.05) , while there were significant differences in the expression of PKD1 and Tyr463-phosphorylated PKD1 among the 4 groups (F= 10.04, 8.27, both P < 0.05) . Additionally, the ALA-PDT group showed significantly lower expression of PKD1 and Tyr463-phosphorylated PKD1 compared with the control group, ALA group and PDT group (all P < 0.05) . Conclusion PKD1 may be involved in the photochemical process of A431 cell apoptosis induced by ALA-PDT, and may promote the occurrence of squamous cell carcinoma by Tyr463 phosphorylation. Key words: Photochemotherapy; Aminolevulinic acid; Neoplasms, Squamous Cell; Protein kinases; Primary cell culture; A431 cell

Acknowledgement to Reviewers 2014

Background and aim: Protein kinase D1 (PRKD1), a serine/threonine kinase, regulates cellular processes such as proliferation, apoptosis, migration, and immune responses. Depending on the tumor context, PRKD1 exhibits either oncogenic or tumor-suppressive functions. This study aimed to delineate the role of PRKD1 in cancer progression and assess its diagnostic and prognostic potential across multiple cancer types.Materials and methods: We analyzed PRKD1 expression using Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), and University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) databases, and assessed its prognostic significance via the Kaplan-Meier plotter. Mutation profiles were examined using cBioPortal, while gene-gene and protein-protein interactions were evaluated through GeneMANIA and STRING, respectively. Pathway enrichment was performed using Enrichr. Findings were validated using three GEO datasets: GSE24152, GSE15641, and GSE110224.Results: PRKD1 expression was significantly downregulated in bladder urothelial carcinoma (BLCA), kidney chromophobe (KICH), and rectum adenocarcinoma (READ) (all p < 0.001). Expression levels varied significantly with clinical parameters, including age, gender, race, and tumor stage. Immune infiltration analysis revealed significant associations between PRKD1 expression and immune cell subsets, namely, B cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells in thyroid carcinoma (THCA), stomach adenocarcinoma (STAD), liver hepatocellular carcinoma (LIHC), and kidney renal clear cell carcinoma (KIRC). A notable inverse correlation was observed between PRKD1 and CD8⁺ T cell levels in THCA (p > 0.05). Survival analysis demonstrated that low PRKD1 expression correlated with improved prognosis in STAD, THCA, and LIHC, whereas high expression was favorable in KIRC (p = 0.001). Promoter methylation of PRKD1 was significantly increased in KICH and READ and decreased in BLCA (all p < 0.001), suggesting epigenetic regulation underlies its differential expression.Conclusion: PRKD1 serves as a potential diagnostic biomarker in BLCA, KICH, and READ, and as a prognostic indicator in STAD, THCA, LIHC, and KIRC. Its expression is modulated by epigenetic mechanisms and correlates with immune cell infiltration, underscoring its relevance in tumor immunobiology and potential as a therapeutic target.

Protein kinase D1 (PKD1), one of the protein kinaseD (PKD) family members, plays a prominent role in multiple bio-behaviors of cancer cells. Low pH and hypoxia are unique characteristics of the tumor microenvironment. The aim of this study was to investigate the role and mechanism of PKD1 in regulating metabolism in the human tongue squamous cell carcinoma(TSCC) cell line SCC25 under a hypoxic condition, as well as growth and apoptosis. Here, we found that hypoxia not only induced the expression of HIF-1α, but also induced the expression and activation of PKD1. Moreover, we inhibited the expression of PKD1 by shRNA interference, and the growth of SCC25 cells under hypoxia was significantly decreased, as well as the expression of HIF-1α, while the percentage of apoptotic SCC25 cells was increased. Furthermore, stable silencing of PKD1 in SCC25 cells under a hypoxic condition decreased glucose uptake, lactate production and glycolytic enzyme (GLUT-1 and LDHA) expression, as well as reduced the phosphorylation of p38MAPK. The results revealed that following inhibition of the expression of PKD1 under a hypoxic condition, the growth and metabolism of the SCC25 cells were significantly suppressed. In contrast, when PKD1 was overexpressed in SCC25 cells, the results were completely reversed, except for growth and apoptosis. Taken together, our results demonstrated that PKD1 not only regulates the hypoxic glycolytic metabolism of cancer cells via regulation of the expression of HIF-1α and glycolytic enzymes, but is also involved in the remodeling of the acidic tumor microenvironment. This study suggests that PKD1 may be a potential target for microenvironment-directed tumor biotherapy.