Aditya Ganju

Date of Award

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

Meena Jaggi, Ph.D.

Committee

Stephen W. Behrman, Ph.D. Santosh Kumar, Ph.D. Yi Lu, Ph.D. Murali M. Yallapu, Ph.D

Keywords

EMT, MTA1, PKD1, Prostate Cancer, TRAMP

Abstract

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.

ORCID

http://orcid.org/0000-0003-1258-0105

DOI

10.21007/etd.cghs.2016.0421

Comments

One year embargo expires December 2017.

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