Despite being one of the most well studied tumor suppressors, there is much evidence that once mutated, p53 can actively promote the progression of many cancers. With respect to breast cancer, tumor-derived mutants of p53 have been implicated in survival, chemoresistance, invasion, migration and metastasis (Brosh and Rotter, 2009
). Since mammary tissue architecture is invariably disrupted during breast carcinogenesis, we sought to delineate the phenotypic effects of mutant p53 in breast cancer. This study describes a possible oncogenic role for certain missense mutants of p53 in disrupting acinar morphogenesis of breast cells, explored using a 3D culture system. In addition, we show that mutant p53 elevates expression of many mevalonate pathway genes and flux through the mevalonate pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast cancer cell morphology in 3D culture.
The mevalonate pathway has recently been implicated in multiple aspects of tumorigenesis, including proliferation, survival, invasion and metastasis (Clendening et al., 2010
; Kidera et al., 2010
; Koyuturk et al., 2007
). Competitive inhibitors of the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, collectively known as statins, have been reported to be cancer-protective for certain malignancies, including breast cancer (Ahern et al., 2011
; Blais et al., 2000
; Cauley et al., 2003
). Yet the cancer-protective effects of statins are not without debate (Baigent et al., 2005
; Browning and Martin, 2007
). Statins have already been employed in multiple preclinical models of breast cancer (Kubatka et al., 2011
; Shibata et al., 2004
). In line with this, we were also able to confirm a significant impact of Simvastatin treatment on MDA-231 breast cancer cells in vivo
It is interesting to note that at least one clinical study investigating the effect of statins in breast cancer noted a subgroup-specific protective effect: specifically, a significantly decreased incidence of hormone receptor-negative (ER−/PR−) tumors was documented in patients takings statins, while no such effect was observed for hormone receptor-positive tumors (Kumar et al., 2008
). Preclinical models, employing either breast cancer cell lines or mouse models of breast cancer, also support a more dramatic role for statins in ER−/PR− breast cancers (Campbell et al., 2006
; Garwood et al., 2010
). Since the majority of breast tumors that bear p53 mutations most commonly are also immunohistochemically classified as ER−/PR− (Sorlie et al., 2001
), it is tempting to speculate that the observed anti-tumorigenic effects of statins are a consequence of mutant p53’s upregulation of the mevalonate pathway.
Gene expression profiling of breast cancers has identified specific subtypes with important clinical, biologic and therapeutic implications (Perou et al., 2000
). Using these expression signatures, most p53 mutations cluster in the basal-like subgroup of breast cancers, which has the poorest prognosis and is notoriously difficult to treat (Sorlie et al., 2001
). Fascinatingly, using a combination of expression signatures and data from over 40,000 compounds screened in the NCI-60 cell lines, three FDA-approved drugs were predicted to be most effective for treating basal-like breast cancers, two of which, Simvastatin and Lovastatin, are inhibitors of HMG-CoA reductase (Mori et al., 2009
). It will be exciting to examine whether stratifying breast cancer patients based on their p53 mutational status can resolve the apparent discrepancies within the rich body of literature linking statins and cancer.
Although we have implicated the mevalonate pathway in the phenotypic effects of mutant p53, it will be of great interest to further delineate the metabolite(s) as well as the downstream signaling pathways that are responsible for these phenotypic effects. While we have demonstrated that metabolic flux through the mevalonate pathway is necessary to maintain the malignant state, with a specific reliance on geranylgeranylation, we cannot rule out the possibility that one or more other metabolites are involved in the phenotypic effects that we observe in 3D culture. If geranylgeranyl pyrophosphate is in fact the key metabolite, it will be very interesting to delineate the geranylgeranylated protein target(s) that mediate the oncogenic effects of the mevalonate pathway in breast cancer cells in 3D culture.
It is interesting to note that, in addition to the mevalonate pathway, a number of fatty acid biosynthesis genes were also significantly affected by mutant p53 depletion from breast cancer cells in 3D culture ( and S6E
). Intriguingly, this is the other major pathway controlled by the SREBP family of transcription factors (Horton et al., 2002
). While much of our data points to a role for SREBP proteins in the regulation of the mevalonate pathway by mutant p53, a direct link is yet to be established. This regulation is likely to occur through one or more of the SREBP proteins, but we cannot rule out the possibility that another factor or factors may also be involved. Mutant p53 may interact directly with elements in the promoters of the sterol biosynthesis genes or alternatively be recruited by a known mutant p53 interacting partner such as NF-Y, SP1, Ets-1 or VDR, which have been shown to recruit mutant p53 to their cognate binding sites (Brosh and Rotter, 2009
; Stambolsky et al., 2010
A number of scenarios have been proposed to explain why human tumors select for mutations in p53. First, mutant p53 may simply be selected for due to loss of wild-type p53 tumor suppressive activity. Second, mutant p53 may acquire neomorphic (i.e. novel gain-of-function) activities which promote tumor growth, many of which have actually been shown to be diametrically opposed to those performed by wild-type p53 (Peart and Prives, 2006
; Stambolsky et al., 2010
). In line with this hypothesis are the findings that Stearoyl-CoA desaturase (encoded by SCD
) is a repression target of wild-type p53 (Mirza et al., 2003
) and that wild-type p53 can suppress a subset of SREBP target genes in a mouse model of obesity (Yahagi et al., 2003
). As the pro-survival roles of wild-type p53 are becoming more apparent (Kim et al., 2009
), a third scenario can be envisaged in which mutant p53 may retain and exaggerate certain wild-type p53 functions, while selectively losing certain tumor suppressive mechanisms such as the ability to induce cell cycle arrest and apoptosis. Regarding the latter two hypotheses, it will be interesting for future studies to examine whether wild-type p53 and/or its family members (p63 and p73) serve to repress sterol biosynthesis genes. Alternatively, the maintenance of high levels of sterol biosynthesis genes by mutant p53 may be a remnant of an unrecognized wild-type p53 function.
These speculations raise another important consideration, that not all p53 mutations are equivalent. Genetic alterations in p53 are often grouped into two classes based on the type of mutant p53 that they produce. Contact mutants, exemplified by p53-R273H, involve mutation of residues that are directly involved in protein-DNA contacts. Conformational mutants, typified by p53-R175H, result in structural distortions in the p53 protein. Our findings that a subset of the sterol biosynthesis genes are significantly higher in large cohorts of human breast tumors bearing mutant p53 suggests that the ability of mutant p53 to upregulate the sterol biosynthesis genes is not constrained to a single class of mutations; however, it will be very interesting for follow-up studies to examine which tumor-derived mutants of p53 can regulate the levels of sterol biosynthesis genes.
In summary, our results demonstrate that mutant p53 can disrupt mammary acinar morphology and that downregulation of mutant p53 from malignant cells is sufficient to phenotypically revert these cells. Here we propose one mechanism, the upregulation of the mevalonate pathway, although one or more additional pathways may play a role. Specifically, we demonstrate that mutant p53 is recruited to the promoters of many sterol biosynthesis genes leading to their upregulation. We hypothesize that tumors bearing p53 mutations evolve to become highly reliant on metabolic flux through the mevalonate pathway, making them particularly sensitive to inhibition of this pathway. At a clinical level, inhibition of the mevalonate pathway, either alone or in combination with other therapies, may offer a novel, and much needed, therapeutic option for tumors bearing mutant p53.