Suppressive Roles of Calreticulin in Prostate Cancer Growth and Metastasis

Calreticulin is an essential, multifunctional Ca²⁺-binding protein that participates in the regulation of intracellular Ca²⁺ homeostasis, cell adhesion, and chaperoning. Calreticulin is abundantly expressed and regulated by androgens in prostate epithelial cells. Given the importance of both calreticulin in multiple essential cellular activities and androgens in prostate cancer, we investigated the possibility of a role for calreticulin in prostate cancer progression. Immunohistochemistry revealed the down-regulation of calreticulin in a subset of human prostate cancer specimens. Prostate cancer cells overexpressing exogenous calreticulin produced fewer colonies in both monolayer culture and soft agar. Furthermore, calreticulin overexpression also inhibited tumor growth in the orthotopic PC3 xenograft tumor model and macroscopic lung metastasis in the rat Dunning AT3.1 prostate tumor model. To address the potential mechanism of calreticulin suppression of prostate cancer, we generated calreticulin mutants with different functional domains deleted. The calreticulin mutants containing the P-domain, which binds to other endoplasmic reticulum chaperone proteins, were sufficient for the suppression of PC3 growth in colony formation assays. Overall, our data support the hypothesis that calreticulin inhibits growth and/or metastasis of prostate cancer cells and that this suppression requires the P-domain.Prostate cancer was the most frequently diagnosed non-skin cancer and the third leading cause of cancer death among American men in 2008.¹ Androgens are intimately associated with prostate cancer progression and as such, androgen ablation remains the standard therapy for patients with metastatic prostate cancer.² However, hormone therapy is not curative and the vast majority of treated patients eventually experience disease progression. Elucidating the mechanism of androgen influence on prostate cancer is important as it may facilitate the development of more effective therapies and methods of disease prevention.Androgen action is mediated through the androgen receptor, which controls the expression of androgen-responsive genes.³ As androgen-responsive genes likely play important roles in prostate cancer progression, the characterization of their expression patterns and functions should provide insight into the roles of androgen in disease development. Calreticulin is one of the androgen-responsive genes in the prostate.^4,5 Androgen ablation by castration rapidly down-regulates calreticulin at both the mRNA and protein levels by more than tenfold. In contrast, androgen replacement rapidly restores the expression of calreticulin in the regrowth of the castrated prostate. Northern blot analysis of the tissue-specificity of calreticulin expression in the rat model shows that the most abundant levels occur in the prostate, as compared with the liver, kidney, brain, heart, muscle, and seminal vesicles. In situ hybridization and immunohistochemistry studies demonstrate that prostatic epithelial cells specifically express calreticulin.^5,6 The expression profile and androgen-responsiveness of calreticulin in the prostate indicate that calreticulin may play a key role in androgen action in prostate epithelial cells.Calreticulin performs a variety of functions within the cell. This evolutionarily conserved protein localizes to the endoplasmic reticulum (ER),^7,8,9 but possibly may also be found at the cell surface.^10,11 Within the ER, calreticulin serves as a molecular chaperone to ensure proper folding of glycoproteins.^12,13,14,15 In addition, calreticulin modulates intracellular Ca ²⁺ homeostasis by its ability to bind Ca²⁺ with high affinity.^6,16,17,18 Other possible activities include integrin α-binding and cell adhesion,^19,20,21,22 major histocompatibility class I assembly,²³ steroid-mediated gene regulation,^24,25,26 as well as Zn²⁺ binding and storage.²⁷ Gene knockout experiments further underscore the importance of this protein, as loss of calreticulin results in embryonic death from defective cardiac development.²⁸ Calreticulin consists of three distinctive domains. The N-domain (residues 1 to 180) is thought to bind heavy metals (Zn²⁺) and interact with other ER chaperones, nuclear receptors, and nucleic acids.²⁹ The P-domain (residues 181 to 290) contains a proline-rich region that forms an extended arm structure and interacts with other chaperones in the lumen of the ER. Lastly, the C-domain (residues 291 to 400) is a highly acidic region that binds Ca²⁺ and is involved in Ca²⁺ storage.³⁰Although the role of calreticulin in normal cellular functions and during embryogenesis is well-established, its role in human carcinogenesis remains poorly understood.³¹ In the current study, we demonstrate the down-regulation of calreticulin protein in a subset of human prostate cancer specimens. Furthermore, we show that calreticulin overexpression in prostate cancer cells inhibits prostate tumor growth and metastasis and that its growth inhibitory role requires the P-domain. Overall, our present study provides evidence, for the first time, that calreticulin is capable of suppressing prostate cancer progression.