There is an evident and urgent need to develop targeted therapeutic strategies against triple-negative breast cancer. To identify these therapeutic targets, a better understanding of the biology of triple-negative breast cancer is therefore needed. In this study, we investigated the biology of triple-negative cancers and identified that MYC signaling is elevated in these tumors. Prior studies have examined MYC expression in breast cancers; however, we find, for the first time, that the expression of multiple MAX binding partners, which can regulate MYC activity, is altered in triple-negative tumors and may therefore contribute to MYC pathway activity.
Several studies have shown that the basal breast cancer subtype exhibits enrichment for a MYC transcriptional gene signature (Alles et al., 2009
; Chandriani et al., 2009
; Gatza et al., 2010
). However, basal breast tumors account for only ~70% of triple-negative tumors, and the importance of MYC signaling in the remaining 30% has previously been unknown. In clinical practice, tumors are routinely evaluated for estrogen receptor, progesterone receptor, and HER2 receptor status and, thus, the pathological determination of the triple-negative subtype is more clinically relevant for deciding on a course of treatment. We found that 33 of 36 triple-negative tumors (92%) in the I-SPY TRIAL had a high or intermediate MYC gene signature score () that correlated with worse outcome (). The present study also demonstrates, for the first time, that MYC signaling is associated with diminished disease-free survival in patients whose tumors exhibited poor response to neoadjuvant chemotherapy ().
To determine if elevated MYC signaling can be exploited to treat triple-negative breast cancer, we assessed the utility of a synthetic-lethal approach between MYC up-regulation and CDK inhibition. Small molecule inhibition of CDK activity was effective at inducing significant cell death in triple-negative cell lines with elevated MYC expression as well as in mouse xenograft models. We found that the mechanism of such cell death included the up-regulation of the proapoptotic BCL-2 family member BIM. Thus, this study represents a significant step forward in identifying apoptotic mechanisms for treating triple-negative breast cancers.
Although prior studies have focused on the many biological functions of MYC, how this information could be translated to develop novel ways to treat breast cancer remains unclear. In an emerging era of personalized medicine, it is crucial to identify what specific patient populations would most benefit from a given treatment strategy. We found that, for patients who experienced a limited tumor response to conventional neoadjuvant chemotherapy, disease-free survival was strongly influenced by MYC expression. Insight into components of MYC signaling, therefore, will provide potential targets that could be exploited as new therapies for this subset of breast cancer patients. However, despite up-regulation of MYC signaling in a variety of human cancers, the potential utility of direct MYC inhibition remains unclear. For instance, MYC knockdown via RNAi has not been found to diminish the viability of cultured tumor-derived cells (Guan et al., 2007
), consistent with our observations in this study with breast cancer cells. In contrast, inhibition of MYC-MAX dimerization using an experimental compound, 10058-F4, led to the induction of apoptosis in cultured human leukemia cells (Huang et al., 2006
). Furthermore, in vivo inhibition of MYC transcriptional activity using a conditional dominant-negative mutant induced cell death and caused tumor regression in a KRAS-initiated mouse lung tumor model (Soucek et al., 2008
). Whether the differences among these observations are a result of the methods used (i.e., knocking down MYC protein expression versus inhibition of its transcriptional activity) or to the extent that each cancer type depends on deregulated MYC activity requires further investigation.
An alternative approach to directly inhibiting MYC is to use a MYC-dependent synthetic-lethal strategy. We previously identified a form of synthetic-lethal interaction between MYC overexpression and CDK1 inhibition using engineered cell lines as well as in vivo model systems (Goga et al., 2007
). More recent studies have shown that RNAi-mediated or small molecule inhibition of two additional cell cycle kinases, CDK2 and aurora kinase B, respectively, has synthetic-lethal interactions with MYC overexpression in certain cancer cell types (Molenaar et al., 2009
; Yang et al., 2010
). Another study has also reported that agonist-mediated activation of a TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) receptor (DR5) induces MYC-dependent synthetic lethality (Wang et al., 2004
). These distinct forms of synthetic lethality appear to require fundamentally different cellular mechanisms of cell death, including different requirements for an intact p53 tumor suppressor pathway (Wang et al., 2004
; Goga et al., 2007
; Molenaar et al., 2009
; Yang et al., 2010
). For example, 44–82% of primary basal breast tumors either lack p53 or harbor p53 mutant alleles (Sørlie et al., 2001
; Carey et al., 2006
). Therefore, CDK1-MYC synthetic lethality, which is p53 independent (Goga et al., 2007
), may be particularly useful in treating these tumors.
The idea of targeting cell cycle kinases to selectively kill tumor cells, which often exhibit higher proliferation rates than nontumorigenic cells, is appealing and has indeed led to the clinical development of several small-molecule CDK inhibitors (Shapiro, 2006
; Malumbres et al., 2008
). However, there have been several issues associated with their clinical development. Previous generations of CDK inhibitors generally suffered from low in vivo potency and poor PK/PD properties. More recent third-generation CDK inhibitors, including dinaciclib, exhibit significantly improved PK/PD properties and offer greater promise for in vivo use. Indeed, dinaciclib was well tolerated in phase I trials and is currently being evaluated in phase II trials against various tumor types (Dickson and Schwartz, 2009
; Parry et al., 2010
). Prior clinical studies have also suffered from a lack of understanding of which tumor types are the most likely to be responsive to CDK inhibitors (Malumbres et al., 2008
). Therefore, selection of patient cohorts based on molecular targets, such as MYC overexpression, may improve the therapeutic potential of CDK inhibitors in clinical trials.
In the present study, we found that CDK inhibition increases BIM protein levels not only in the model cells engineered to overexpress MYC but also in a panel of patient-derived triple-negative breast cancer cell lines with elevated MYC expression. Elevated BIM plays a direct role in CDK inhibition–induced cell death. Prior studies have found that BIM isoforms can be regulated at both the transcriptional and posttranslational levels. Protein expression of the shortest isoform Bim-S is sufficient to potently induce apoptosis (O’Connor et al., 1998
). In contrast, the activity of the longest isoform BIM-EL can also be modulated by distinct phosphorylation mechanisms that can either stabilize the protein or induce its degradation via the proteasome pathway (Hübner et al., 2008
). In our studies, we found that both isoforms can be up-regulated in epithelial cells after CDK inhibitor treatment.
Previous studies have shown that BIM expression is elevated upon MYC overexpression (Egle et al., 2004
; Hemann et al., 2005
). Having uncovered that CDK inhibition triggers induction of BIM expression, we became interested in studying the relationship between the protein expression levels of MYC and BIM in a panel of untreated breast cancer cell lines. We did not find a correlation between MYC and BIM expression in these cells regardless of their molecular subtypes or receptor status (unpublished data). Therefore, in a given cellular context, it is likely that the relative increase in BIM activity in response to CDK inhibition, not the absolute basal level of BIM expression, determines whether or not apoptosis can be initiated. Thus, the protein stoichiometry among BIM and antiapoptotic BCL-2 family members such as BCL-2, BCL-xL, and MCL-1 is likely to dictate if apoptosis is triggered. In this respect, a combinatorial approach of CDK inhibition with inhibition of antiapoptotic BCL-2 family members would be predicted to have a synergistic effect in inducing cell death in MYC overexpressing triple-negative tumors. Indeed, several BH3 mimetics (Chonghaile and Letai, 2008
), namely ABT-737/263 (Oltersdorf et al., 2005
) and obatoclax (Nguyen et al., 2007
), are currently under development for clinical use.
In conclusion, we have shown utility for small molecule CDK inhibitors in the treatment of triple-negative breast tumors with elevated MYC expression. It is likely that CDK inhibitors will be effective not only against MYC-overexpressing triple-negative tumors but also for aggressive receptor-positive tumors with elevated MYC expression (i.e., luminal B). Methods for immunohistochemical analysis on paraffin-embedded primary tumor biopsies for MYC protein expression have been previously described (Gurel et al., 2008
; Ruzinova et al., 2010
). In addition, gene expression profiling methods on primary breast tumors are becoming available for use in the clinic (van ’t Veer et al., 2002). Such technologies could be applied to assess whether MYC signaling is elevated in patient tumor samples (Alles et al., 2009
; Chandriani et al., 2009
; Gatza et al., 2010
). Thus, these detection methods for MYC activity have the potential to be translated for routine use in the clinic. Considering the lack of established targeted therapeutics against triple-negative tumors, we propose that MYC status will prove useful as a predictive biomarker of response to CDK inhibitors for the treatment of triple-negative breast cancers.