BERKELEY, CA (UroToday.com) - Prostate cancer is the most prevalent non-cutaneous type of cancer in men. Although efforts to treat localized, primary prostate tumors have been met with considerable success, metastatic prostate cancer remains essentially incurable. As metastasis is the major morbidity and mortality factor for patients with prostate cancer, progress towards elucidating the mechanism of metastasis will offer rational ways of intervention with great benefit for the patient in the clinic.
Cancer metastasis is multi-step process in which tumor cells acquire progressively multiple traits, including detachment from the extracellular matrix, anoikis resistance (defined as resistance to a special type of cell death triggered when they lose adhesion to extracellular matrix), migration and invasion through the basement membrane, intravasation to blood and lymphatic vessels, extravasation from the circulation to distant sites, the ability to stimulate angiogenesis, and, finally, formation of macroscopic metastases. At the molecular level, a number of signaling pathways, including integrins and Akt, contribute to the survival and progression of a tumor. Integrins are heterodimeric αβ transmembrane receptors that connect the extracellular matrix to the cytoskeleton and play important roles in migration, invasion, and anoikis resistance. In particular, β1 integrin (ITGB1) is known to confer higher survival and metastatic capacity to a number of cancer cells, including those of prostate origin. Activation of the phosphati¬dylinositol 3′ kinase (PI3K) pathway, due mainly to loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene, occurs frequently in human prostate cancer. The serine/threonine protein kinase Akt is a downstream effector of PI3K and an important regulator of various cellular functions, including cell metabolism, transcription, survival and proliferation. Cancer cells rely heavily on active Akt to survive from a number of insults such as genotoxic stress, growth factor depletion, and metastasis.
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. Rad9 is involved in almost all aspects of DNA repair, including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination. However, Rad9 can interact with several other proteins outside the context of the 9-1-1 complex and checkpoint functions. Aberrant Rad9 expression has been associated with prostate, 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. 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.
The impetus for the current study was the observation that immunohistochemical analysis of normal and tumor prostate specimens showed that Rad9 expression increased along with cancer progression stages, suggesting a role for Rad9 in prostate malignant progression. Thus, 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 first showed that suppressing the expression of Rad9 protein, by RNA interference, reduced both migration and invasion of DU145 and PC3 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 DU145 cells.
Malignant cells have developed mechanisms to evade anoikis and either proliferate without matrix support or enter quiescence until a more suitable environment is presented. Anoikis resistance is, therefore, a prerequisite of tumor metastasis and is considered a hallmark of cancer. Akt kinase plays a pivotal role in the resistance of malignant cells to anoikis. Our results show that Rad9 down-regulation impaired Akt phosphorylation when DU145 and LNCaP cells were maintained in suspension. Conversely, when Mrad9 was ectopically expressed in DU145 with reduced levels of endogenous Rad9, Akt phosphorylation was restored, and cells became more resistant to anoikis.
Finally, we discovered that silencing of Rad9 leads to a marked down-regulation of integrin β1. Conversely, ectopic expression of Mrad9 restores integrin β1 levels when endogenous Rad9 expression is knocked down in DU145 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 integrin β1.
Having established the role of Rad9 in migration and invasion in vitro, we are now directing our efforts to elucidate the ability of Rad9 to impact on metastasis in vivo. The information gained will help define new molecular events in carcinogenesis and could potentially reveal targets for novel anti-cancer therapies.
Written by:
Constantinos G. Broustas‡ and Howard B. Lieberman§ as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
‡Center for Radiological Research, Columbia University College of Physicians and Surgeons, New York, NY 10032 USA
§Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY 10032 USA
Rad9 contributes to prostate tumor progression by promoting cell migration and anoikis resistance - Abstract
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