The p53 status can influence the role of Sam68 in tumorigenesis

The expression and activities of RNA binding proteins are frequently dysregulated in human cancer. Their roles, however, appears to be complex, with reports indicating both pro-tumorigenic and tumor suppressive functions. Here we show, using two classical mouse cancer models, that the role of KH-type RNA binding protein, Sam68, in tumor development can be influenced by the status of the p53 tumor suppressor. We demonstrate that in mice expressing wild type p53, Sam68-deficiency resulted in a higher incidence and malignancy of carcinogen-induced tumors, suggesting a tumor suppressive role for Sam68. In marked contrast, Sam68-haploinsufficiency significantly delayed the onset of tumors in mice lacking p53 and prolonged their survival, indicating that Sam68 accelerates the development of p53-deficient tumors. These findings provide considerable insight into a previously unknown relationship between Sam68 and the p53 tumor suppressor in tumorigenesis.

To further investigate the physiological roles of Sam68, a mouse model was generated by homologous recombination [17]. These whole body knockout mice are born with no visible deformities and live to old age [17]. Phenotypically, Sam68-null mice are lean and are protected against age-related bone loss [17,18]. These mice also have significant motor coordination defects attributed to RNA metabolism dysregulation in neurons [19][20][21][22]. Interestingly, crossbreeding with spinal muscular atrophy (SMA∆7) mice rescued the body weight and viability of SMA∆7, suggesting that the loss of Sam68 could be a therapeutic avenue for some patients with spinal muscular atrophy [23]. Sam68-null mice do not develop spontaneous tumors, demonstrating that Sam68 is not a bona fide tumor suppressor like p53 [24,25]. However, Sam68-haploinsufficiency delays the onset of MMTV-PyMT-driven mammary tumors and reduces dissemination of lung metastasis, implicating that Sam68 is required for mammary tumorigenesis [26].
As a prototypic member of the STAR proteins, Sam68 has signaling properties and functions downstream of Src family kinases to promote cell survival [26,27]. In addition, Sam68 regulates cell cycle progression and

Research Paper
apoptosis. Specifically, Sam68 modulates transcription and mRNA translation, and functions as a major regulator of pre-mRNA alternative splicing [2]. It has been demonstrated that Sam68 depletion by siRNA promotes cell cycle arrest, and decreases the proliferation of many cancer cell lines [4,7,8,15]. In particular, Sam68 has been shown to enhance the inclusion of CD44 variable exon 5 and the splicing of cyclin D1b isoform [28,29], both of which are known to promote cell proliferation. Depletion of Sam68 from breast cancer cell lines upregulates cyclindependent kinase inhibitors p21 and p27, while also reducing Akt phosphorylation [4]. Moreover, Sam68 contributes to the protection of prostate cancer cells from genotoxic agents [15]. Hence, these findings strongly suggest Sam68 as an RNA binding protein required for the proliferation of certain cancer cells.
Sam68 has also been shown to harbor tumor suppressive functions, but this activity is not well understood, nor is there any evidence of this tumor suppressive role in vivo. It has been proposed that the loss of Sam68 leads to NIH3T3 cell transformation; however, this was not rescued by Sam68, suggesting that additional events were responsible for transformation in this report [30]. Consistent with its tumor suppressor-like activities, the overexpression of Sam68 in murine fibroblasts induces cell cycle arrest and apoptosis [31]. Moreover, Sam68 expression favors alternative splicing of the pro-apoptotic Bcl-x(s) isoform, which further demonstrates its tumor suppressor-like activity [32,33].
The development of mouse cancer models has greatly advanced our understanding of the mechanisms underlying tumor initiation, progression, metastasis, and acquired chemoresistance [34][35][36]. Specifically, the chemical carcinogen-based inducible tumor models are essential tools for investigating tumor development in a controlled manner [34,35]. For instance, the azoxymethane (AOM)-inducible mouse model provides a reproducible system for studying spontaneous colon cancer, where tumors develop frequently in the distal part of the colon, and exhibit features similar to spontaneous colon cancer found in humans [37]. Additionally, genetically engineered mice, such as the p53-deficient mice [38], have been particularly valuable for the identification of molecular mechanisms and pathways affected by p53-deficiency, as the loss or mutation of TP53 occurs in approximately 50% of all cancers [25,39]. Indeed, p53-deficient mice are predisposed to a range of spontaneous tumors, including lymphomas and sarcomas [40,41]. Recently, we have identified Sam68 as a transcriptional co-activator of p53 [42], suggesting that Sam68 may also have tumor suppressor activities like p53.
In this study, we report the role of Sam68 in tumorigenesis using both p53-proficient and p53-deficient mouse models of cancer. We show that in p53 wild type mice, Sam68-deficiency enhanced AOM-induced colon tumorigenesis, whereas in p53-deficient mice, Sam68-haploinsufficiency delayed the onset of spontaneous tumors. Hence, we demonstrate for the first time that p53 status can influence the role of Sam68 during tumorigenesis.

DISCUSSION
In the present study, we examined the physiological role of Sam68 in tumor development using two different mouse cancer models. For Sam68-proficient and deficient mice in a p53 wild type FVB background, we implemented the azoxymethane (AOM)-inducible colon carcinogenesis model. We observed that Sam68-deficient (p53 +/+ ;Sam68 −/− ) mice displayed higher tumor burden with increased malignancy than wild type controls (p53 +/+ ;Sam68 +/+ ), demonstrating that Sam68 has tumor suppressor-like activities in vivo. We also generated p53-null mice expressing Sam68 (p53 −/− ;Sam68 +/+ ) or haploinsufficient dose of Sam68 (p53 −/− ;Sam68 +/− ). In this p53 −/− C57BL/6 background, we observed that Sam68 haploinsufficiency caused significant delays in the onset of spontaneous tumors compared to Sam68 wild type mice. These findings show that in the absence of p53, Sam68 can exhibit pro-tumorigenic properties. Collectively, our results demonstrate that p53 seems to act as a switch to influence whether Sam68 slows or accelerates tumor development. Taken together, these findings provide insight into a previously unknown relation between Sam68 and the p53 tumor suppressor in tumorigenesis.
RNA binding proteins are becoming increasingly appreciated as fundamental players in human cancers [45,46]. Like tumor suppressors, abnormal expression or mutations in RNA binding proteins have been shown to alter their function, and many are associated with a cancer phenotype [46]. We posit for the first time that Sam68-deficiency leads to increased tumorigenesis, where p53 +/+ ;Sam68 −/− mice displayed significantly higher AOM-induced tumor burden and malignancy compared to p53 +/+ ;Sam68 +/+ mice. Therefore, in this context, Sam68 behaves as a tumor suppressor. Indeed, Sam68 has been shown to be pro-apoptotic mainly by regulating Bcl-x(s) splicing [31][32][33]. Moreover, the fact that Sam68 functions as a co-activator of p53 is consistent with its tumor suppressor role [42]. Interestingly, it has been shown that p53 does not influence the rate of tumor initiation, but instead is required to prevent the malignant progression of tumors [47], which parallels the phenotype of the AOM mouse model. However, untreated Sam68 −/− mice are not predisposed to spontaneous tumor development [17], similar to the phenotypes of p21 −/− and p21 −/− ;Puma −/− ;Noxa −/− mice [48,49]. This perhaps explains why, as observed with the p21 gene, that no known mutations of Sam68 have been found in human tumors [50]. Furthermore, unlike p53, the function of Sam68 may be partially redundant with other STAR family RNA binding proteins, such as Sam68 paralogs SLM-1 and SLM-2 [51,52].
Given the interplay between Sam68 and p53, it is almost expected that Sam68 would lose its tumor suppressive function when p53 is depleted. Indeed, studies in p53 −/− mice showed that decreased Sam68 expression significantly delayed the onset of spontaneous tumors, indicating a pro-tumorigenic role for Sam68 in this mouse model. Fu et al. recently reported that Sam68 lessens colon tumor development in Apc min716/+ mice via regulation of the PARP1-NF-κB-anti-apoptotic gene axis [53]. Furthermore, Sam68-deficient human colon cancer cells are sensitive to genotoxic stress-induced apoptosis [53].
Although it is uncertain how p53 loss or inactivation can contribute to the pro-tumorigenic function of Sam68, a large body of literature has elucidated molecular mechanisms by which Sam68 can support tumor growth.
As a STAR family protein, Sam68 can link signal transduction with RNA processing to modulate cancerrelevant splicing events that enhance cell survival and proliferation [1][2][3]. For instance, Fyn-mediated tyrosine phosphorylation of Sam68 promotes splicing of the antiapoptotic Bcl-x(L) isoform in prostate and pancreatic  incidence in p53 +/+ ; Sam68 −/− , p53 −/− ; Sam68 +/+ , and p53 −/− ; Sam68 +/− C57BL/6 mice. Tumor formation was monitored over a period of 10 months, and sacrificed at first clinical signs of discomfort (p < 0.0001). www.impactjournals.com/oncotarget cancer cells [32,54]. Additionally, Sam68 has been identified as a downstream target of the MAPK ERK1/2 pathway, where serine/threonine phosphorylation of Sam68 enhances tumorigenic CD44 variable exon 5 inclusion [28]. Similarly, activation of the MAPK pathway also stimulates Sam68-mediated splicing of cyclin D1b isoform to favor cell proliferation [29]. The involvement of Sam68 in signaling during tumorigenesis has also been observed previously in a MMTV-PyMT mouse model, where Sam68-haploinsufficiency delayed mammary tumorigenesis [26], consistent with the phenotype of our p53 −/− ;Sam68 +/− mice. Of note, mammary tumor cells derived from the MMTV-PyMT mice are p53 wild type and are sensitive to p53 gene therapy, but the antitumor mechanisms remain elusive [55]. Furthermore, Sam68 may promote tumorigenesis by taking part in the transcription machinery. In prostate cancer cells, Sam68 functions as a transcriptional co-activator of the androgen receptor (AR) [14], a nuclear hormone receptor driving the onset and progression of prostate cancer [56]. Sam68 also modulates the promoter specificity of NF-κB to induce CD25 expression implicated in tumorigenesis [57]. Additionally, Sam68 is an essential component of the MLL oncogenic transcriptional complex by acting as a bridging molecule for MLL and PRMT1, and the knockdown of Sam68 suppressed MLL-mediated transformation [58]. In further support of its pro-tumorigenic properties, Sam68 overexpression may drive tumor progression by downregulating tumor suppressive miRNAs, including miR-203 and miR-204 [13,16].
Sam68 overexpression, phosphorylation, and its cytoplasmic localization have been associated with a significant risk factor for poor prognosis [8,9,59]. It is also known that extensive posttranslational modifications such as phosphorylation can influence its RNA binding activity [28,32,60]. For example, the elevated expression, mislocalization, and tyrosine phosphorylation of Sam68 all contribute to poor prognosis in breast cancer patients [4,59]. Consistently, aberrant tyrosine phosphorylation and cytoplasmic localization of Sam68 has also been observed in various other cancer cells [8,9,26,61,62]. Together, the evidence suggests that aberrant Sam68 regulation, including sequestration of Sam68 from its nuclear role or inactivation by phosphorylation contributes to exacerbate tumorigenesis.
Although further studies are required to understand the mechanism underlying p53's ability to switch the functional outcome of Sam68, we have illustrated the importance of defining the molecular contexts in which targeting RNA binding proteins may be beneficial for anticancer therapy. Much remains to be defined on how RNA binding proteins and their regulatory networks contribute to tumor initiation and progression.

Mice breeding, AOM treatment, and colon tumor preparations
All animal procedures followed the Canadian Council on Animal Care guidelines and were approved by the McGill Animal Care Committee. Age-matched (10 to 12 weeks) p53 +/+ ;Sam68 +/+ and p53 +/+ ; Sam68 −/− mice in FVB background (cohorts of 15 in each group) were injected intraperitoneally once a week for 8 weeks with 10 mg/kg of body weight azoxymethane (AOM) (Sigma-Aldrich) dissolved in saline. Animals were systematically examined twice per week for the appearance of clinical symptoms. Animals showing signs of discomfort such as weight loss, rectal bleed, and prolapse were sacrificed. End points were defined as 13 weeks after the first injection. After sacrificing the mice, entire colons were removed, rinsed with PBS, opened longitudinally and fixed flat on strips of 4% paraformaldehyde-soaked Whatman filter paper. Colons were assessed in a blinded fashion under a stereo-dissecting microscope as previously described [63]. Tumors were measured using a clear transparency of 1 mm 2 graph paper, and the total surface area was determined based on the total number of squares overlaying the tumor, as described [64].
The p53 −/− mice (Catalog# 002101) were obtained from JAX laboratories. Both Sam68 −/− and p53 −/− mice were maintained on a C57BL/6 background and crossbred. Animals showing signs of discomfort such as weight loss were sacrificed.

Statistical analysis
Statistical analysis was performed using Student's t test with the GraphPad software. A p-value of < 0.05 was considered to be statistically significant.