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The mammalian Cks family consists of 2 well-conserved small proteins, Cks1 and Cks2. Cks1 has been shown to promote cell-cycle progression by triggering degradation of p27kip1. The function of Cks2 in somatic mammalian cells is not well understood although it is required for the first metaphase/anaphase transition during the meiosis. Emerging evidence shows that elevated expression of Cks1 and Cks2 is often found in a variety of tumors, and is correlated with poor survival rate of the patients. Here we demonstrated that expression of Cks1 and Cks2 were elevated in prostate tumors of human and animal models, as well as prostatic cancer cell lines. Forced expression of Cks1 and Cks2 in benign prostate tumor epithelial cells promoted cell population growth. Knockdown of Cks1 expression in malignant prostate tumor cells inhibited proliferation, anchorage-independent growth, and migration activities, whereas knockdown of Cks2 expression induced programmed cell death and inhibited the tumorigenicity. Collectively, the data suggest that elevated expression of Cks1 contributes to the tumorigenicity of prostate tumor cells by promoting cell growth and elevated expression of Cks2 protects the cells from apoptosis. Thus, the finding suggests a novel therapeutic strategy for prostatic cancer based on inhibiting Cks1 and Cks2 activity. © 2008 Wiley-Liss, Inc.
The human Cks (also designated as CksHs) family consists of 2 well-conserved members, Cks1 and Cks2, both of which are identified based on the sequence homology to yeast suc1 and Cks1 (Cdc28 kinase subunit 1) that are essential for cell cycle-control.1–3 Emerging evidence shows that the 2 Cks members in mammalian cells have distinct regulatory function from the yeast counterparts. Cks1 is required for SCFSkp2-mediated ubiquitination and degradation of p27kip1, which is essential for the G1/S transition during the cell cycle.4, 5 Although function of Cks2 in the cell-cycle is not clear, expression of both Cks1 and Cks2 has been shown oscillates during the cell cycle and is positively related to cell proliferation.6 Recently, Cks2 has been shown essential for the first metaphase/anaphase transition of mammalian meiosis.7
Numerous reports demonstrate that expression of Cks2 is frequently elevated in tumors of different tissue origins, including nasopharyngeal carcinoma, melanocytic tumors, Wilms tumor, breast, bladder, cervical, esophageal, lymphoid and metastatic colon cancer.6, 8–14 In addition, expression of Cks2 is downregulated by p53, a tumor suppressor, at the transcription and the protein levels.15 Similarly, elevated expression of Cks1 has been found in tumors from a variety of tissue origins, and is correlated with poor survival rate of oral squamous cell carcinoma.16–23 Knockdown of Cks1 inhibits growth and tumorigenicity of oral squamous cells.23 Consistent with the finding that Cks1 is a negative regulator of a cell cycle control protein, p27kip1, elevated expression of Cks1 is found coincident with the reduction of p27kip1 proteins in tumor cells. Expression of p27kip1 is often aberrantly reduced in cancer cells, including prostate cancer cells. We recently reported that the fibroblast growth factor (FGF) signaling axis directly regulates activity of Cks1 during the G1/S transition in the cell cycle through FGF receptor substrate 2α (FRS2α), a proximal FGF receptor-interactive adaptor protein of the FGF receptor tyrosine kinase, which connects multiple downstream signaling molecules to the FGF receptor tyrosine kinase.24
The prostate is an accessory organ of the male reproductive system, which consists of epithelial and stromal compartments separated by basement membranes. Cancers arising from the prostate epithelium are the most commonly diagnosed cancer and the second most common cause of cancer death in American males. In America alone, about 230,000 new cases and 30,000 deaths are expected every year. To date, whether the Cks family is overexpressed in prostate tumor, and if yes, whether aberrantly expressed Cks contributes to prostate tumor initiation and progression remain to be characterized. Recently, it has been shown that Cks1 expression is associated with the aggressive behavior of prostate cancer.25 In addition, treating LNCaP prostate cancer cells with an herbal mixture, PC-SPES, inhibits cell proliferation and reduces expression of Cks2.26
To determine whether overexpression of the Cks family was associated with prostate tumorigenesis, we analyzed the expression pattern of Cks1 and Cks2 in the Dunning prostate tumor model of rats, the TRAMP prostate cancer model of mice, human prostate cancer cell lines, and human prostate cancer samples. The results showed that in comparison with normal prostate tissues that only weakly express Cks1 and Cks2, all tested human, rat and mouse prostate tumor tissues and cells exhibited elevated expression of Cks1 and Cks2. Forced expression of Cks1 and Cks2 by transfection in benign prostate tumor cells somewhat promoted cell population increases. Consistently, knockdown of Cks1 and Cks2 expression by shRNA in malignant AT3 cells inhibited cell population growth. In addition, knockdown of Cks1 expression also inhibited anchorage-independent growth, and migration activities, and knockdown of Cks2 expression induced the cells to undergo massive program cell death, especially when the cells were maintained in suboptimal growth condition. The results suggested that elevated expressed Cks1 may contribute to prostate tumorigenesis by promoting proliferation, anchorage-independent growth and migration of the cells, and Cks2 by protecting cells from undergoing programmed cell death. The finding also provides new hints for cancer therapeutic strategies based on inhibition of the Cks activity.
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Here we demonstrate that expression of Cks1 and Cks2 was elevated in prostate tumors, including the Dunning prostate tumors in rat, the TRAMP tumors in mouse, and more importantly, in human prostate tumors and cell lines. Forced expression of Cks1 and Cks2 in benign prostate tumor epithelial cells accelerated cell growth; knockdown of Cks1 and Cks2 malignant prostate tumor cells inhibited cell growth, anchorage-independent growth and migration activity. In addition, knockdown of Cks2 expression induced apoptosis in vitro and compromised tumorigenic activity of the cells in vivo. The results suggest that elevated expression of Cks1 may contribute to tumorigenicity of prostate tumor cells by promoting cell growth and elevated expression of Cks2 by protecting the cells from undergoing apoptosis.
The function of the Cks family appears to be complicated. In yeast, both Cks1 and Cks2 bind to Cdk1 at a high affinity and function as a suppressor of Cdk1 mutants.1, 3, 34 Despite the high sequence homology between the 2 members, whether Cks1 and Cks2 in mammalian cells elicit redundant or synergistic activities is not well understood although 1 report demonstrates that Cks1 compensates for Cks2 loss of function in the germ line.7 In mammalian cells, Cks1 induces an allosteric change in Skp2, which increases its affinity for phosphorylated p27kip1 and triggers ubiquitination of p27kip1, resulting in a rapid proteasome-mediated degradation of p27kip1 during G1/S transition in the cell cycle. To date, decreases in p27kip1 expression or mutations of p27kip1 has been reported in tumors of many tissue origins, including prostate cancer.35, 36 Here we showed that forced expression of Cks1 reduced p27kip1 in the cells and accelerated cell growth, while knockdown Cks1 expression in malignant AT3 cells inhibited proliferation and increased p27kip1 abundance (Fig. 2).
In addition to being a mitotic inhibitor, p27kip1 also has cell cycle-independent function, including regulating cell migration. However, p27kip1 has been shown both promoting and inhibiting cell migration.37–42 Recently, it has been showed that the binding of p27kip1 to the microtubule-destabilizing protein stathmin regulates the activity of p27kip1 to repress cell migration. Upregulation of p27kip1 or downregulation of stathmin expression results in inhibition of cell motility that is essential for invasion and metastasis of tumors.43, 44 Our data showed that knockdown of Cks1 expression increased p27kip1 abundance in the cells and inhibited cell migration, which was consistent with the finding that p27kip1 inhibits migration and invasion of tumor cells. In addition, inhibition of p27kip1 in LNCaP cells significantly increases colony formation in soft agar, suggesting that p27kip1 inhibits anchorage-independent growth activity of prostate cancer cells.45 Consistently, our data showed that knockdown of Cks1 expression increased abundance of p27kip1 and inhibited colony formation activity of the malignant AT3 cells. The result further suggests that overexpression of Cks1 might contribute to the tumorigenicity through promoting anchorage-independent growth activity.
In contrast to Cks1, knockdown of Cks2 expression in AT3 cells did not increase p27kip1 abundance. Instead, it slightly reduced the abundance of p27kip1. It is possible that the reduction of p27kip1 abundance in AT3 cells was a result of cell death caused by knockdown of Cks2 expression (Fig. 2). Further experiments are needed to address the issue. Similarly, forced expression of Cks2 did not reduce p27kip1albeit it accelerated DTE cell growth (Fig. 2). Therefore, although changes in Cks2 expression affect cell growth and has been shown to oscillate during the cell cycle,6 it is likely that Cks2 may not directly control the cell cycle progression. Indeed, expression of Cks2 in DU145, AT3 and TRAMP C2 cells was not reduced when the cells were cultured in serum-free medium for 48 hr (Supplemental Fig. 1), in which condition, the cell proliferation was relatively lower than in full culture medium. It is possible that changes in Cks2 expression may impact cell growth by preventing apoptosis. Both cell growth and apoptosis assays showed that knockdown of Cks2 expression in malignant AT3 cells had a more potent impact in cells maintained in nonoptimal culture conditions than in those maintained in optimal condition. This may explain why the leaky expression of Cks2 shRNA had limited effects on stock cell cultures that were maintained in the optimal conditions, yet, had significant effects on in vivo tumorigenesis analyses where the cells were implanted subcutaneously and did not have optimal supplies of oxygen and nutrition.
Together with the fact that Cks2 null mice did not have apparent defect in all tissues except the testes,7 our data here imply that Cks2 may not be essential for growth, differentiation and maintenance of normal prostatic cells, yet, is essential for protecting the cells from undergoing apoptosis in harsh growth conditions, such as deficient in nutritional or oxygen supplies. Thus, inhibition of Cks2 activity may be of therapeutic potential for tumor treatment, and promotion of Cks2 activity may be useful for preventing cells to undergo apoptosis because of insufficient oxygen or nutrition supplies, such as in stroke, heart attacks or other injury. In addition, although having distinct roles, the 2 Cks members may synergistically contributed to prostate tumorigenesis by promoting cell proliferation and preventing apoptosis.
In summary, here we report that aberrant expression of Cks1 in prostate tumor cells promoted tumorigenicity by promoting proliferation, anchorage-independent growth and cellular migration activity, while aberrant expression of Cks2 may promote the tumorigenicity by protecting the cells from apoptosis. Together with the reports that Cks1 and Cks2 are not essential for somatic cell growth and tissue homeostasis, the results here suggest a novel strategy for prostatic cancer treatment without affecting normal somatic cells.