REGULATION OF CANCER METASTASIS BY PROTEIN KINASE D1: A GLOBAL REGULATORY CASCADE

Protein Kinase D1 (PKD1) is a serine threonine kinase which is downregulated in Prostate, Breast and Colon Cancer. It functions as a tumor suppressor in different cancer cells. Downregulation of PKD1 is known to be associated with aggressiveness of the cancer. PKD1 is known to regulate many key oncogenic signaling pathways such as E-cadherin, β-catenin and Androgen Receptor signaling pathways. Aberrant expression of these oncogenic pathways leads to transformation of cells from normal to malignant phenotype, thereby leading to increased proliferation, growth and metastasis to distant organs of these cancer cells. Literature evidence also points to the fact that E-cadherin β-catenin and PKD1 play a role in regulation of epithelial mesenchymal transition (EMT). To fully understand how PKD1 regulates β-catenin signaling, we investigated the effect of PKD1 overexpression on β-catenin signaling in colon cancer cells. We observed that PKD1 overexpression is responsible for inhibition of cell proliferation and colony formation ability of different colon cancer cell lines. Moreover, nuclear PKD1 overexpression leads to inhibition of β-catenin transcription activity in colon cancer cells. Further evaluation in in vivo mouse model showed that PKD1 is responsible for inhibition of colon cancer tumor growth in xenograft mouse model. This paved way for us to look for the effect of PKD1 on other downstream targets of β-catenin pathway which regulate EMT process in cancer cells such as Metastasis associated Protein 1. Metastasis associated Protein 1 (MTA1) is a nucleosome remodeling and histone deacetylase protein (NuRD) which is overexpressed in all the cancers. MTA1 is an initiator of epithelial and mesenchymal transition and is responsible for cancer cells metastasizing to different organs of the body. Expression of MTA1 directly correlates with the aggressiveness of the cancer. MTA1 is known to regulate β-catenin and Androgen Receptor signaling pathways leading to cancer cells acquiring metastatic capabilities. Therefore, in our study we evaluated the inverse correlation between MTA1 and PKD1 in different cancer cells. To investigate the cellular effect of PKD1 in prostate and colon cancer, stable PKD1 overexpressing prostate (C4-2) and colon cancer cells (SW480) were utilized. PKD1 overexpression inhibited MTA1 expression in prostate and colon cancer cells. PKD1 interacts, phosphorylate, translocate and degrades MTA1. Kinase domain and N terminal domain of PKD1 play a significant role in MTA1 interaction and phosphorylation. Phosphorylation of MTA1 leads to nuclear export via golgi and trans-golgi network to lysosome. Bryostatin-1 is a macrocyclic lactone which modulates PKD1 activity. Bryostatin-1 was used to activate PKD1 expression in C4-2 cells and MTA1 translocation was then tracked. This translocation of MTA1 to lysosome is a ubiquitin dependent phenomenon leading protein degradation. PKD1 overexpression leads to inhibition of tumor growth and bone metastasis leading to inhibition of osteoblast to osteoclast formation as determined by RANK expression. PTEN Knockout and TRAMP mouse model also show inverse correlation between PKD1 and MTA1 expression in prostate tissues at different weeks. Human tissue microarray of prostate, colon and breast cancer (MTA1 is overexpressed and PKD1 is downregulated in breast cancer, therefore, we tested our hypothesis in breast cancer as well) showed inverse correlation between PKD1 and MTA1 in different grade tumor tissue signifying clinical relevance of this correlation. For proof of concept of our hypothesis we used ormeloxifene because Bryostatin-1 has mild toxicity issue. Ormeloxifene is a novel modulator of PKD1 activity and it targets rapidly dividing cells Further, we investigated the effect of ormeloxifene on activation of PKD1 leading to inhibition of cancer metastasis. We observed specific activation of PKD1 expression of ormeloxifene which inhibited MTA1 expression leading to inhibition of tumor growth in xenograft mouse. We further evaluated the efficacy of ormeloxifene to inhibit metastatic prostate cancer cells (PC3 and DU145). Ormeloxifene showed excellent anti-cancer efficacy against prostate cancer as it inhibited cell proliferation, invasion and migration of metastatic prostate cancer cells. Moreoever, ormeloxifene induced cell cycle arrest at G0/G1 phase by regulating key cell cycle regulatory proteins. It also inhibited metastasis of prostate cancer leading to inhibition of key metastatic markers involved to epithelial mesenchymal transition. Ormeloxifene also showed excellent in vivo efficacy against metastatic prostate cancer cells. Therefore, ormeloxifene could be a potential therapeutic modality for metastatic cancers as it targets EMT signaling. To conclude, we for the very first time have elucidated a novel regulatory mechanism of PKD1 mediated regulation of MTA1 that plays an important role in cancer progression and metastasis. For cancer cells to metastasize PKD1 expression is suppressed with subsequent increased expression of MTA1. We elucidated that repression of MTA1 with subsequent activation of MTA1 leads to attenuation of cancer metastasis. Moreover, therapeutic modality that targets this novel regulatory pathway leading to activation of PKD1 and inhibition of MTA1 is an ideal candidate for treatment of advanced stage metastatic cancers

miRNA nanotherapeutics for cancer

miRNAs are noncoding RNA molecules that regulate gene expression through diverse mechanisms. Increasing evidence suggests that miRNA-based therapies, either restoring or repressing miRNA expression and activity, hold great promise. However, the efficient delivery of miRNAs to target tissues is a major challenge in the transition of miRNA therapy to the clinic. Cationic polymers or viral vectors are efficient delivery agents but their systemic toxicity and immunogenicity limit their clinical usage. Efficient targeting and sustained release of miRNAs/ anti-miRNAs using nanoparticles (NPs) conjugated with antibodies and/or peptides could reduce the required therapeutic dosage while minimizing systemic and cellular toxicity. Given their importance in clinical oncology, here we focus on the development of miRNA nanoformulations to achieve enhanced cellular uptake, bioavailability, and accumulation at the tumor site

Molecular Insights into Targeting PKD1 for Prostate Cancer Treatment

Background: Prostate cancer has a poor prognosis due to late diagnosis and ineffective multimodal clinical treatment. Efforts are underway to create strategies for resolving the abnormal expression of molecular targets implicated in disease development and progression. We previously reported that the serine threonine kinase Protein Kinase D1 (PKD1) regulates a multitude of tumor suppressor functions, including cell aggregation, motility, proliferation, and invasion in prostate cancer. Thus, PKD1 is regarded as a promising therapeutic target for the treatment of prostate cancer. Objective: The goal of this study was to investigate the therapeutic potential of ormeloxifene (ORM), a pharmacological modulator with well-defined PK/PD and safety profiles in humans, for PKD1 restoration in prostate cancer. Methods: The anticancer effect of ORM on PKD1 and associated signaling mechanisms in prostate cancer was investigated using proliferation, clonogenicity, migration, invasion, western blotting, and qPCR analysis. Results: In comparison to the vehicle-treated group, ORM treatment decreased prostate cancer cell proliferation, invasion, migration, and colony formation in a dose-dependent manner. In C4-2 cells, ORM treatment selectively induces PKD1 expression at both the mRNA and protein levels. Furthermore, our findings revealed that ORM efficiently suppresses MTA1 expression in prostate cancer cells. MTA1 physically interacts with PKD1 and has been shown to have an inverse correlation with it. Our results also showed that ORM treatment enhances the therapeutic efficacy of decetaxel. Conclusion: Taken together, these findings show that ORM has anticancer properties in prostate cancer via restoring PKD1

Pharmacological restoration of PKD1: A novel strategy for prostate cancer therapy

Background: Prostate cancer has poor prognosis owing to late diagnosis and ineffective multimodal clinical treatment. Extensive efforts are ongoing to establish methods that can resolve the expression of genes implicated in disease development and treatment. Previously, we reported that Protein Kinase D1 (PKD1), a serine threonine kinase, controls a number of tumor suppressor functions including cell aggregation, cell motility, cell proliferation, and cell invasion. Thus, PKD1 is considered as an emerging therapeutic target for prostate cancer treatment. Objective: To investigate the restoration of PKD1 by a pharmacological modulator ormeloxifene, which showed well-defined PK/PD and safety profiles in humans. Methods: Proliferation, clonogenicity, migration, invasion, western blotting and qPCR analysis were performed to investigate the anticancer effect of ORM, docetaxel and/or their combination on PKD1 and related signaling mechanisms in prostate cancer. Results: ORM treatment inhibited cell proliferation, invasion, migration and colony formation abilities of prostate cancer cells in a dose-dependent manner compared to vehicle treated group. ORM treatment selectively induces the expression of PKD1 both at mRNA and protein levels in C4-2 cells. Moreover, our results have also shown that ORM effectively attenuates MTA1 expression in prostate cancer cells. MTA1 physically interact and shown to have inverse relationship with PKD1. In addition, we observed that ORM treatment enhances the therapeutic efficacy of docetaxel in C4-2 cells. Our results also indicate that ORM treatment potentiate the effects of docetaxel as determined by MTS and colony formation assays. Conclusion: These results suggest that ORM exhibit potent anticancer activity via restoration of PKD1 in prostate cancer

Protein kinase D1 regulates subcellular localisation and metastatic function of metastasis-associated protein 1

Background: Cancer progression and metastasis is profoundly influenced by protein kinase D1 (PKD1) and metastasis-associated protein 1 (MTA1) in addition to other pathways. However, the nature of regulatory relationship between the PKD1 and MTA1, and its resulting impact on cancer metastasis remains unknown. Here we present evidence to establish that PKD1 is an upstream regulatory kinase of MTA1. Methods: Protein and mRNA expression of MTA1 in PKD1-overexpressing cells were determined using western blotting and reverse-transcription quantitative real-time PCR. Immunoprecipitation and proximity ligation assay (PLA) were used to determine the interaction between PKD1 and MTA1. PKD1-mediated nucleo-cytoplasmic export and polyubiquitin-dependent proteosomal degradation was determined using immunostaining. The correlation between PKD1 and MTA1 was determined using intra-tibial, subcutaneous xenograft, PTEN-knockout (PTEN-KO) and transgenic adenocarcinoma of mouse prostate (TRAMP) mouse models, as well as human cancer tissues. Results: We found that MTA1 is a PKD1-interacting substrate, and that PKD1 phosphorylates MTA1, supports its nucleus-to-cytoplasmic redistribution and utilises its N-terminal and kinase domains to effectively inhibit the levels of MTA1 via polyubiquitin-dependent proteosomal degradation. PKD1-mediated downregulation of MTA1 was accompanied by a significant suppression of prostate cancer progression and metastasis in physiologically relevant spontaneous tumour models. Accordingly, progression of human prostate tumours to increased invasiveness was also accompanied by decreased and increased levels of PKD1 and MTA1, respectively. Conclusions: Overall, this study, for the first time, establishes that PKD1 is an upstream regulatory kinase of MTA1 status and its associated metastatic activity, and that the PKD1-MTA1 axis could be targeted for anti-cancer strategies

Cucurbitacin D Reprograms Glucose Metabolic Network in Prostate Cancer

Prostate cancer (PrCa) metastasis is the major cause of mortality and morbidity among men. Metastatic PrCa cells are typically adopted for aberrant glucose metabolism. Thus, chemophores that reprogram altered glucose metabolic machinery in cancer cells can be useful agent for the repression of PrCa metastasis. Herein, we report that cucurbitacin D (Cuc D) effectively inhibits glucose uptake and lactate production in metastatic PrCa cells via modulating glucose metabolism. This metabolic shift by Cuc D was correlated with decreased expression of GLUT1 by its direct binding as suggested by its proficient molecular docking (binding energy -8.5 kcal/mol). Cuc D treatment also altered the expression of key oncogenic proteins and miR-132 that are known to be involved in glucose metabolism. Cuc D (0.1 to 1 µM) treatment inhibited tumorigenic and metastatic potential of human PrCa cells via inducing apoptosis and cell cycle arrest in G2/M phase. Cuc D treatment also showed inhibition of tumor growth in PrCa xenograft mouse model with concomitant decrease in the expression of GLUT1, PCNA and restoration of miR-132. These results suggest that Cuc D is a novel modulator of glucose metabolism and could be a promising therapeutic modality for the attenuation of PrCa metastasis

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer-stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2-dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non-cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells

Cucurbitacin D Reprograms Glucose Metabolic Network in Prostate Cancer

Prostate cancer (PrCa) metastasis is the major cause of mortality and morbidity among men. Metastatic PrCa cells are typically adopted for aberrant glucose metabolism. Thus, chemophores that reprogram altered glucose metabolic machinery in cancer cells can be useful agent for the repression of PrCa metastasis. Herein, we report that cucurbitacin D (Cuc D) effectively inhibits glucose uptake and lactate production in metastatic PrCa cells via modulating glucose metabolism. This metabolic shift by Cuc D was correlated with decreased expression of GLUT1 by its direct binding as suggested by its proficient molecular docking (binding energy −8.5 kcal/mol). Cuc D treatment also altered the expression of key oncogenic proteins and miR-132 that are known to be involved in glucose metabolism. Cuc D (0.1 to 1 µM) treatment inhibited tumorigenic and metastatic potential of human PrCa cells via inducing apoptosis and cell cycle arrest in G2/M phase. Cuc D treatment also showed inhibition of tumor growth in PrCa xenograft mouse model with concomitant decrease in the expression of GLUT1, PCNA and restoration of miR-132. These results suggest that Cuc D is a novel modulator of glucose metabolism and could be a promising therapeutic modality for the attenuation of PrCa metastasis