Metastasis is the major morbidity and mortality factor for men with prostate cancer. Patients routinely receive androgen-deprivation therapy (ADT); however, almost all of these men develop resistance to ADT and progress to metastatic castration-resistant prostate cancer (mCRPC). Therefore, elucidating the mechanism of metastasis will offer rational ways of intervention with great benefit for the patient in the clinic.
Advanced prostate cancer is characterized by frequent genomic aberrations in the androgen receptor (AR), phosphatidylinositol 3′ kinase (PI3K), and DNA repair signaling pathways. Global gene expression analysis has recently revealed that aberrations of ATM, BRCA1, and BRCA2 in mCRPC are observed at substantially higher frequencies compared with those in primary prostate cancers. Furthermore, three out of four mCRPC tumors exhibit hypermutation and harbor alterations in the mismatch repair pathway genes MLH1 and MSH2.
The observation that DNA repair genes are upregulated in mCRPC, as well as in primary tumors predicted to metastasize, may indicate that elevated DNA repair efficiency is associated with increased risk of distant metastasis. However, accumulating evidence suggests that some DNA repair genes, including DNA-PKcs, PARP-1, BRCA1, and RAD9, can promote metastasis by multiple means not necessarily related to their DNA repair capacity. Thus, DNA-PKcs plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. It contributes to metastasis development by stimulating angiogenesis, migration and invasion. Furthermore, DNA-PKcs acts as a transcription factor and synergizes with AR to promote mCRPC. PARP-1 has also been shown to be critical for AR transcriptional function and maintenance of AR-dependent cancer phenotypes, including tumor progression. PARP-1 enzymatic activity is significantly upregulated in mCRPC, and promotes both AR chromatin binding and transcription factor functions. Interestingly, ETS transcription factors, which are aberrantly expressed in approximately 50% of prostate tumors, induce a transcriptional program that includes several invasion-associated genes (e.g., EZH2). ETS gene-mediated transcription and the resulting cell invasion, intravasation, and metastasis are dependent on DNA-PKcs and PARP-1 activity.
RAD9 protein has an established role in the DNA damage response and DNA repair. As part of the RAD9-HUS1-RAD1 complex, it acts as a sensor of DNA damage that enables ATR kinase to phosphorylate and activate its downstream effector CHK1. In addition, RAD9 promotes base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination. Moreover, human RAD9 can function as a sequence-specific transcription factor. Specifically, RAD9 can bind to p53 DNA-binding consensus sequences in the promoter region of p21Waf1/Cip1 and NEIL1, and enhance transcription of these genes.
Aberrant RAD9 expression has been associated with breast, lung, skin, thyroid, and gastric cancers. We have shown previously that RAD9 is overexpressed in human prostate cancer specimens as well as prostate cancer cell lines. In addition, RAD9 expression can confer radioresistance to prostate cancer cells. However, RAD9 also correlates with poor prognosis independent of damage induction in numerous tumor types. Most importantly, down-regulation of RAD9 in human tumor cell line xenografts impairs growth in nude mice, thus establishing a causative role for RAD9 in prostate cancer. Furthermore, immunohistochemical analysis of non-cancer and tumor prostate specimens showed that RAD9 expression increased along with cancer progression stages, suggesting a role for RAD9 in prostate malignant progression.
In a recent article in The Journal of Biological Chemistry, we examined a number of in vitro metastasis markers such as cell motility, invasion, anoikis resistance and anchorage-independent growth, as well as activation of tumor promoting signaling pathways, specifically integrin expression and AKT activation. We showed that suppressing the expression of RAD9 protein, by RNA interference, reduced both migration and invasion of human prostate cancer cell lines, whereas ectopically re-expressing Mrad9, the mouse homolog of human RAD9, restored the phenotype in these cells. Likewise, anchorage-independent growth, which reflects most faithfully the in vivo metastatic potential of a cancer cell, was impaired when RAD9 was silenced in prostate cancer cells.
Resistance to anoikis, that is, the induction of programmed cell death upon cell matrix detachment, is a prerequisite of tumor metastasis and a hallmark of cancer. The AKT kinase signaling pathway confers protection to malignant cells against anoikis. Our results showed that RAD9 down-regulation impaired AKT phosphorylation when prostate cancer cells were maintained in suspension. Conversely, when Mrad9 was ectopically expressed in cells with reduced levels of endogenous RAD9, AKT phosphorylation was restored, and cells became more resistant to anoikis.
ITGB1 is known to confer higher survival and metastatic capacity to a number of cancer cells, including those of prostate origin. We discovered that silencing of RAD9 leads to a marked down-regulation of integrin β1 (ITGB1), whereas ectopic expression of Mrad9 restores ITGB1 levels when endogenous RAD9 expression is knocked down in DU145 prostate cancer cells. Furthermore, reduction of ITGB1 protein levels by a specific siRNA negated the effect of Mrad9 on migration and invasion, suggesting that RAD9 affects these processes through the activity of ITGB1.
In conclusion, our findings indicate an important role for RAD9 in prostate cancer progression and metastasis. They also reveal that RAD9, like other DNA repair proteins, display pleiotropic functions. Elucidating which of these functions are important to prostate tumor progression could potentially reveal targets for novel anti-cancer therapies.
Written by: Constantinos G. Broustas and Howard B. Lieberman
Broustas CG, Zhu A, Lieberman HB. (2012). Rad9 protein contributes to prostate tumor progression by promoting cell migration and anoikis resistance. J Biol Chem. 287(49):41324-33.
Broustas CG, Lieberman HB. (2014). DNA damage response genes and the development of cancer metastasis. Radiat Res. 181(2):111-30.
Broustas CG, Lieberman HB. (2014). RAD9 enhances radioresistance of human prostate cancer cells through regulation of ITGB1 protein levels. Prostate. 74(14):1359-70.