Opposing roles for mammary epithelial-specific PPARγ signaling and activation during breast tumour progression

Given recent evidence implicating a protective role for PPARγ in breast cancer [12,13,14], the MG cell-specific contribution of this receptor was evaluated during DMBA-induced breast tumourigenesis using PPARγ-MG KO and PPARγ-WT mice. Cotreatment with a gold standard PPARγ activator, ROSI, further provided the ability to identify PPARγ-dependent anti-breast tumour progression signaling pathways specific to MG cells. Other groups have examined the MG-specific contribution of PPARγ in breast cancer, using overexpression [18] and dominant negative knockout [19] approaches that only target the PPARγ1 isoform. Here, the Cre-loxP system was used to delete expression of both PPARγ protein isoforms, and thus, eliminate any confounding compensatory effects. In addition, the ROSI dose and regimen used here was previously shown to effectively activate PPARγ signaling [20-22] and achieve serum glucose profiles within human therapeutic ranges in mice [23,24]. Surprisingly, it was discovered that PPARγ-MG KO mice are protected more so than PPARγ-WTs during DMBA-mediated breast tumourigenesis; whereas, PPARγ activation by ROSI rescues PPARγ-WTs but renders PPARγ-MG KOs more susceptible to breast tumour progression. These findings suggest that PPARγ expression within MG cells may be a strong candidate biomarker for identifying patient populations with aggressive breast tumours, as well as aid in predicting patients likely to benefit from novel chemotherapeutic use of PPARγ activating drugs.

The findings that PPARγ-MG KO mice respond more favourably, for example in OS, than PPARγ-WTs following tumourigenic initiation by DMBA, but do worse following cotreatment with a PPARγ activating ligand were unexpected. These surprising outcomes may be explained, at least in part, by the increased total mammary tumour and malignant mammary tumour incidences, and decreased mammary tumour latency, that were observed in DMBA + ROSI-treated PPARγ-MG KOs compared to those treated with DMBA alone. Collectively, these findings suggest that PPARγ expression in MG cells and ROSI activation in mice lacking MG-specific PPARγ is potentially harmful during chemical-mediated breast tumour progression. Given that ROSI activation produced detrimental effects exclusive to knockout mice suggests that PPARγ-independent effects of this drug may be partly responsible [25]. These interesting observations underscore the importance of personalized medicine, and the need for characterizing normal breast and mammary tumour epithelial expression of PPARγ before considering TZD-like drugs as chemotherapeutic strategies for breast cancer patients. ROSI may still represent a viable chemotherapeutic option if expression of MG cell-specific PPARγ remains intact.

With respect to the mouse mammary tumour outcomes observed in these studies, both MG-specific PPARγ deficiency and activation produced similar results. Although paradoxical, these comparable outcomes may reflect similar signaling pathways resulting from cofactor mobility. For example, in the PPARγ-MG KO model, coactivators and/or corepressors normally bound by the PPARγ/RXRα complex may be released to interact with their downstream signaling targets and exert their intended effects similar to when PPARγ is activated. This may partially explain why DMBA Only-treated PPARγ-MG KOs have better OS compared to DMBA Only-treated PPARγ-WTs, but comparable to PPARγ-WTs treated with DMBA + ROSI. A similar mechanism has been reported in a PPARβ KO mouse model, whereby an antiinflammatory corepressor (Bcl-6) is free to exert its effects in both PPARβ-deficient and PPARβ-activated cell contexts [26]. RNAseq and ChIPseq assays evaluating global PPARγ/RXRα interactions with specific cofactors and gene targets would help clarify if this mechanism is involved in the context of breast tumourigenesis, but is beyond the scope of these studies.

MG-specific expression of PPARγ and BRCA1 were confirmed in untreated PPARγ-WT but abolished in PPARγ-MG KO mammary glands. Importantly, ROSI cotreatment increased PPARγ expression in mammary-derived lymphomas and carcinomas from PPARγ-WT and PPARγ-MG KO mice, but only specifically augmented BRCA1 in PPARγ-WTs. BRCA1 is a critical tumour suppressor gene that possesses a PPRE within its promoter region [17]. We have previously demonstrated that BRCA1 expression can be upregulated in fat cells via adipocyte-specific PPARγ activation [13]. Accordingly, the current study provides similar evidence that BRCA1 is a target of PPARγ in MG cells. This specific interaction may contribute to the improved outcomes observed among DMBA + ROSI-treated PPARγ-WT mice, via BRCA1-mediated DNA damage repair and/or blocking aromatase-dependent estrogen production [27].

Cox-2 is a key PG-synthesizing enzyme and a breast cancer prognostic marker of poor outcome [28]. Consequently, Cox-2 protein expression is observed in many epithelial tumours, including breast cancer [29], with increasing levels associated with advanced tumour grade [30,31]. ROSI cotreatment repressed Cox-2 in PPARγ-WT tumours, but dramatically amplified it in PPARγ-MG KOs. This marked increase in Cox-2 protein levels among DMBA + ROSI-treated PPARγ-MG KO tumours may partially explain the poor survival and mammary tumour outcomes within this study group. Indeed, Cox-2 promotes aromatase transcription [32] and renders cells resistant to apoptosis and even chemotherapy [31]; however, some of these properties may be mediated by PG levels. That we did not observe any significant differences with respect to serum PGE levels in any group provides evidence that other PG products, or perhaps even Cox-2 activity independent of PG production, may be involved in this setting and requires further study.

Although the Cox-2 gene contains a PPRE within its promoter [33,34], PPARγ-dependent and PPARγ-independent mechanisms both positively and negatively regulate Cox-2 gene transcription depending on cell- and stimulus-specific contexts [33,35,36,37]. Given Cox-2 expression was lower in DMBA + ROSI-treated PPARγ-WT tumours suggests MG cell-specific PPARγ activation may play a role in suppressing Cox-2 protein levels, which is similar to our findings with respect to mammary secretory epithelial-PPARγ [14]. On the other hand, DMBA + ROSI-treated PPARγ-MG KO tumours showed a dramatic increase in Cox-2 protein levels suggesting that a PPARγ-independent process is likely responsible. It has been demonstrated that PPARγ ligands activate Cox-2 transcription via receptor-independent stimulation of the MAPK-NF-κB pathway [38,39]. Moreover, PPARγ-independent activation of the glucocorticoid receptor by ROSI may also be responsible for increased Cox-2 gene expression [40,41], although this remain to be proven.

The PTEN gene promoter also reportedly contains a PPRE [33,34], and so it was not surprising that PTEN protein levels were markedly reduced among PPARγ-MG KO mammary tumours in the DMBA Only group. Interestingly, DMBA + ROSI-treated PPARγ-WT mice had reduced PTEN expression among mammary tumours than observed in mammary tumours from respective DMBA Only controls. This may be reflective of the decreased mammary tumour progression in DMBA + ROSI-treated PPARγ-WT mice. Taken together, these data suggest PPARγ is required for normal PTEN expression in malignant mammary tumours, but PTEN is not an early PPARγ downstream signaling target in benign mammary tumours, and may be a fruitful area for research in future studies.

Variable PPARγ protein levels were observed among mammary tumours from PPARγ-WT and PPARγ-MG KO by Western blot analysis. Among PPARγ-WT tumours, the variable pattern may reflect alternative pathways acquired during tumourigenic progression of initiated cells, some of which may silence PPARγ expression. There are indeed reports that PPARγ levels decline as human breast tissue becomes increasingly malignant [42], which is consistent with our hypothesis of its role as a suppressor of breast tumour progression. Alternatively, the inherent cellular heterogeneity of these mammary tumours, that likely contain differing amounts of PPARγ expressing stromal adipocytes, endothelial cells and immune cells, may contribute to the observed variability. Although a possible mosaic expression pattern of the MMTV promoter [43] cannot be discounted, differing percentages of stromal PPARγ expressing cells may also explain the variability of PPARγ expression observed in PPARγ-MG KO mammary tumours. This is supported by our IF data showing specificity and extent of PPARγ deletion among mammary epithelial cells of untreated PPARγ-MG KO mice. It is also possible that other non-mammary epithelial cell sources of PPARγ signaling may have contributed to the outcomes of these in vivo tumourigenesis studies. We previously showed that mammary adipocyte-specific PPARγ blocks breast tumour progression in part via upregulation of BRCA1 [13]. Interestingly, in the present study, treatment with ROSI caused induction of BRCA1 expression in PPARγ-WT, but not PPARγ-MG KO mouse mammary tumours. This suggests activation of PPARγ may protect against breast tumour progression only when mammary epithelial-stromal crosstalk contains functional PPARγ signaling in both cell types, and is the focus of additional studies beyond the scope of this work.

Untreated knockout serum contained lower levels of known proinflammatory and chemotactic cytokines, including eotaxin, IFN-γ and MCP-1α [44-46], as well as other contextually-dependent proinflammatory signals, such as IL-4 and GM-CSF [45], that could have possibly rendered PPARγ-MG KOs less susceptible to breast cancer compared to PPARγ-WTs when challenged with DMBA. DMBA + ROSI cotreatment also rescued PPARγ-WTs via downregulation of serum VEGF. This may be the result of direct PPARγ activity via a PPRE in the VEGF promoter [47], or indirectly via other PPARγ targets such as BRCA1, which can silence VEGF expression and secretion [48], or Cox-2, which can induce VEGF expression [49].

Irrespective of treatment, all PPARγ-MG KOs exhibited significantly lower levels of serum IL-4, IL-10, IL-13, eotaxin, GM-CSF, IFN-γ and MIP-1α compared to PPARγ-WT mice. This is particularly intriguing because these cytokines are commonly produced by macrophages and T lymphocytes [44,46,50,51]. Given that this cytokine expression pattern is genotype-specific raises the possibility that PPARγ-MG KO mice possess fewer macrophages and T cells, and thus experience reduced inflammation, compared to PPARγ-WTs. This explanation may provide another layer why knockout mice were less susceptible to breast tumourigenesis when challenged with DMBA.