Dr. Balaji's Laboratory

Urology Research Laboratory

Dr. K.C. Balaji is a urological oncologist and a physician-scientist with a career-long interest in advanced prostate cancer (PC).

Balaji’s laboratory was the first to discover that a novel kinase, Protein Kinase D1 (PrKD), is downregulated in castration-resistant prostate cancer (CRPC)1,2; a lethal and advanced-stage disease in men with PC. Since the discovery over two decades ago, the laboratory has detailed proteins that interact with and/or are phosphorylated by PrKD and contribute to the progression of PC.

Initial studies revealed that PrKD interacts with E-cadherin, a major cell adhesion molecule, which altered cellular aggregation and motility3. Furthermore, PrKD suppressed epithelial-to-mesenchymal transition (EMT) by phosphorylating Snail, a major transcriptional repressor of E-cadherin. Interestingly, E-cadherin in turn altered subcellular distribution and function of PrKD independent of Protein Kinase Cs (PKCs) or diacylglycerol, and that membrane targeting of PrKD is sufficient for activation, which is a novel mechanism of PrKD activation4.

E-cadherin interacts with beta-catenin and maintains cell-cell adhesion and cytoskeletal instability. PrKD interacts and phosphorylates beta-catenin at threonine 120 (T120)5 that links beta to alpha-catenin, which interacts with cytoskeletal actin filaments and maintains cytoskeletal stability. Downregulation of PrKD decreases T120 phosphorylation of beta-catenin, which causes nuclear translocation of beta-catenin and increases transcriptional activity5.

Hypothetically, the lack of T120 phosphorylation can lead to uncoupling alpha to beta-catenin binding at the membrane, causing nuclear translocation of alpha and beta-catenin, where alpha-catenin is known to inhibit beta-catenin transcriptional and DNA damage repair (DDR) activity.

The laboratory also discovered a novel autorepressive signaling loop in which nuclear beta-catenin interacts with MYC/MAX transcription factor at the PrKD promoter site and represses PrKD gene expression6.

The regulation of PrKD expression by MYC/MAX heterodimer is a novel discovery that is distinct from previously known epigenetic regulation PrkD in gastric cancer. This discovery, for the first time, allowed strategies to upregulate the expression of a tumor suppressor such as PrKD by inhibiting the inhibitory transcription factor. The laboratory is currently funded by the Department of Defense, Prostate Cancer Research Program, to develop novel c-Myc inhibitors in the management of PC.

Balaji’s laboratory also discovered that PrKD interacts and influences the function of other proteins such as androgen receptor (AR) and heat shock protein 27 (HSP27), which are known to play a major role in PC7,8.

One of the proteins that interact with the kinase domain of PrKD is metallothionein 2A (MT2A), which is a metal-responsive small protein known to scavenge free radicals and trace metals. Intriguingly, the prostate by far has the highest concentration of zinc (Zn) in the human body compared to any other organ and chelation of Zn reduced MT2A expression associated with increased sensitivity to cisplatin chemotherapy and radiation 9,10.

The findings provide impetus to study increasing radiation sensitivity in PC using chelation strategy11. PrKD was shown to increase the secretion of matrix metalloproteinase-2 and -9, which in contrast to previously described pro-invasive functions in cancer were shown to play an anti-oncogenic role in PC12.

In order to study the PrKD-centric signaling in vivo, the laboratory developed a prostate-specific PrKD knockout (KO) mice model. Whereas germline loss of PrKD is lethal to an embryo, prostate-specific loss of PrKD did not demonstrate discernible phenotypic changes. However, prostatic specific K-RasG12D knock-in mutation or PTEN KO in PrKD KO mice showed prostatic hyperplasia and neoplastic changes respectively13.

In addition, the laboratory generated a novel “Prorainbow” mice consisting of florescent-labeled prostate cells with a characteristic hue that could be used to study clonal origins of cells. The discoveries have led to the development of a PrKD-centric biomarker panel that is able to discriminate in vitro indolent from aggressive prostate cancer cells14.

The laboratory has also developed other study models including 3D microcapsules15 and PC organoids. The studies have identified several genes and proteins for therapeutic targeting in PC, which are involved in a variety of pathways that are critical in PC progression including AR signaling, DNA damage, tumor suppressors and protooncogenes.

The importance of DNA damage genes in metastatic and aggressive PC such as intraductal carcinoma (IDCP) and cribriform variants is being recognized. Overexpression of PrKD in PC cells, like the two other serine-threonine kinases (CHEK1 and CHEK2), mediates cell cycle arrest16. PrKD phosphorylates cell division cyclin phosphatase 25 (CDC25) independent of CHEK kinases and induces cell cycle arrest in the G1/S phase.

These preliminary findings suggest that PrKD mediated cell cycle arrest may be a novel DNA damage response pathway distinct from p53 dependent ATM/CHK2 and p53 independent ATR/CHK1 pathways. The laboratory continues to investigate the role of PrKD and other genes/proteins in the causality of metastatic and aggressive variants of PC.

Bibliography and References

  1. Jaggi M, Rao PS, Smith DJ, Hemstreet GP, Balaji KC. Protein kinase C mu is down-regulated in androgen-independent prostate cancer. Biochem Biophys Res Commun. 2003;307(2):254-260.
  2. Jaggi M, Du C, Zhang W, Balaji KC. Protein kinase D1: a protein of emerging translational interest. Front Biosci. 2007;12:3757-3767.
  3. Jaggi M, Rao PS, Smith DJ, et al. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. 2005;65(2):483-492.
  4. Li Z, Zhang C, Chen L, et al. E-Cadherin Facilitates Protein Kinase D1 Activation and Subcellular Localization. J Cell Physiol. 2016;231(12):2741-2748.
  5. Du C, Jaggi M, Zhang C, Balaji KC. Protein kinase D1-mediated phosphorylation and subcellular local ization of beta-catenin. Cancer Res. 2009;69(3):1117-1124.
  6. Nickkholgh B, Sittadjody S, Rothberg MB, et al. Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer. Oncotarget. 2017;8(45):78811-78824.
  7. Mak P, Jaggi M, Syed V, et al. Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells. Biochem Biophys Res Commun. 2008;373(4):618-623.
  8. Hassan S, Biswas MH, Zhang C, Du C, Balaji KC. Heat shock protein 27 mediates repression of androgen receptor function by protein kinase D1 in prostate cancer cells. Oncogene. 2009;28(49):4386-4396.
  9. Rao PS, Jaggi M, Smith DJ, Hemstreet GP, Balaji KC. Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer. Biochem Biophys Res Commun. 2003;310(3):1032-1038.
  10. Smith DJ, Jaggi M, Zhang W, et al. Metallothioneins and resistance to cisplatin and radiation in prostate cancer. Urology. 2006;67(6):1341-1347.
  11. Gmeiner WH, Boyacioglu O, Stuart CH, Jennings-Gee J, Balaji KC. The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn(2+) -chelation and inhibiting the serine protease Omi/HtrA2. Prostate. 2015;75(4):360-369.
  12. Biswas MH, Du C, Zhang C, Straubhaar J, Languino LR, Balaji KC. Protein kinase D1 inhibits cell proliferation through matrix metalloproteinase-2 and matrix metalloproteinase-9 secretion in prostate cancer. Cancer Res. 2010;70(5):2095-2104.
  13. Fang X, Gyabaah K, Nickkholgh B, Cline JM, Balaji KC. Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate. Prostate. 2015;75(9):988-1000.
  14. NickKholgh B, Fang X, Winters SM, et al. Cell line modeling to study biomarker panel in prostate cancer. Prostate. 2016;76(3):245-258.
  15. Fang X, Sittadjody S, Gyabaah K, Opara EC, Balaji KC. Novel 3D co-culture model for epithelial-stromal cells interaction in prostate cancer. PloS one. 2013;8(9):e75187.
  16. Nickkholgh B, Sittadjody S, Ordonez K, Rothberg MB, Balaji KC. Protein kinase D1 induces G1-phase cell-cycle arrest independent of Checkpoint kinases by phosphorylating Cell Division Cycle Phosphatase 25. Prostate. 2019;79(9):1053-1058.