Homeless Cells Escape Death and Deliver Lethal Cancer.

Breast cancer is the most common cancer in women worldwide. In the United States, 276 480 women were diagnosed with breast cancer and 42 170 patients lost their lives to the disease in 2020. Breast cancer accounts for 15% of cancer-related deaths in women, and at the time of diagnosis approximately 20% of patients exhibit lethal disease, triple-negative breast cancer (TNBC) (1). The incidence of TNBC is significantly higher among premenopausal women and is associated with more aggressive disease, including shorter time to progression, higher rates of metastasis, and poor prognosis compared to other breast cancer subtypes. At the cellular level TNBC is characterized by a lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), a molecular landscape that presents significant therapeutic challenges, and TNBC patients fail to respond to treatments targeting ER, PR, or HER2. Owing to the lack of targeted therapeutics, chemotherapy is currently the only medical intervention proven to increase survival in TNBC patients. Despite initial response to chemotherapy, therapeutic resistance emerges leading to lethal disease. Identification of actionable signaling effectors that mediate apoptosis and metastasis promises novel targets for precision-targeted therapies to improve clinical outcomes in TNBC patients.

The development of resistance in metastatic TNBC is orchestrated by the functional overlapping signaling pathways that regulate cell survival, resistance to anoikis, epithelial-mesenchymal transition (EMT), invasion, endocrine status (including androgen receptor, AR) and angiogenesis. In the new manuscript by Rosas and colleagues, “A positive feedback loop between TGF-β and androgen receptor supports triple-negative breast cancer anoikis resistance” published in Endocrinology (2), the authors identify a positive loop orchestrating a signaling interaction between transforming growth factor-β (TGF-β) (a multifunctional cytokine that regulates all the previously listed processes) and AR (nuclear transcription factor). This cooperation of players takes “no prisoners” of TNBC cells, but on the contrary facilitates their resistance to death via anoikis (2). Anoikis, a distinct mode of programmed cell death that occurs on cell detachment from the extracellular matrix, thus disrupting integrin-ligand interactions, is a critical mechanism in preventing ectopic cell growth or attachment to an inappropriate matrix. Among these “homeless” cell populations, a lack of integrin ligation leads to decreased focal adhesion kinase and integrin-linked kinase activity, which impairs downstream survival signaling with talin as the primary driver. Indeed, a decade ago we first established that talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance (3). Consequently, overcoming tumor cell survival by lifting resistance to anoikis and consequential phenotypic reversion from EMT to mesenchymal-epithelial transition (MET), provides a powerful molecular targeting platform to impair metastasis and overcome therapeutic resistance (4).

TGF-β is a multifunctional cytokine that regulates multiple processes in cancer, serving as a tumor suppressor in the early stage of tumorigenesis and functionally associated as a promoter of aggressive tumor invasion and metastasis in the late stages of tumor progression. The role of TGF-β signaling in promoting tumor progression to metastasis could be through its ability to block anoikis, induce EMT (SMADs), and regulate the actin cytoskeleton organization (cofilin). Resistance to cell death by anoikis is facilitated by EMT and leads to metastasis, as the cancer cells lose their cell-cell adhesions and polarity, adopt a more invasive and migratory mesenchymal phenotype that can survive anoikis, and colonize distant sites. Inhibition of TGF-β signaling may sensitize cancer cells to anoikis by engaging a rigorous phenotypic reprogramming of cancer cells from EMT to MET (5). A limitation of the present study is that the authors did not interrogate (in any depth) the phenotypic landscape of the TNBC cells protected from anoikis by the TGF-β/AR as there was a transition from anchorage-dependence to -independence and TGF-β or AR inhibition. This could determine the downstream ramifications of targeting these pathways in anoikis resistance, by assessing the expression profile of EMT markers such as E- and N-cadherin, vimentin, Slug, and Snail, and anoikis resistance cell survival proteins such as Bcl-2 and talin (3). Nonetheless, one easily recognizes that blockade of the TGF-β signaling mechanism promises a therapeutic value in lifting anoikis resistance and preventing tumor progression to lethal disease. In preclinical mouse models of breast cancer, constitutive expression of a TGF-β antagonist under the control of a mammary gland–specific promoter could prevent the development of metastases with few adverse effects (6). However, as recognized by the authors, clinical trials of TGF-β inhibitors have met with mixed success, so the focus on development of combination therapies to target pathways leading to lethal tumors is the challenge in effectively treating patients with TNBC.

Thus in a “conspiracy” mode, the cytokine TGF-β signals the nuclear receptor AR to synergistically provide full protection of homeless breast cancer cells from dying via anoikis. This is not an “unorthodox” move, because AR is expressed in approximately 50% of TNBC cases, and presents itself as an actionable target for a therapeutic gain. Moreover, the role of AR in promoting an anchorage-independent, cancer stem cell–like population in TNBC has been previously established by the same investigative team. AR overexpression and activity was linked to anoikis resistance in forced suspension culture conditions, and increased the pool of cancer stem cell–like cells. Preclinical models provided further evidence that inhibition of AR could suppress tumor growth (7). Phase 2 clinical trials assessing the efficacy of an AR antagonist (enzalutamide) in metastatic therapeutically resistant TNBC revealed a modest clinical benefit; thus, exploitation of combination therapies with AR antagonists directly targeting the AR functional contribution to anoikis resistance could provide further benefit and increase survival in TNBC patients. In assessing an AR contribution to tumor resistance, one cannot dismiss the possible involvement of constitutive active splice variants of AR, such as AR-V7, commonly associated with emergence of therapeutically resistant prostate cancer. Since AR variants have been detected in advanced TNBC, the present study is limited in the lack of consideration of their role in breast cancer progression and treatment resistance, and toward establishing the therapeutic impact of treatment with second-generation antiandrogens in patients with lethal TNBC.

The translational insights provided by this elegant work on a combinatorial therapeutic strategy targeting TNBC and impairing metastatic spread by using inhibitors of TGF-β and AR signaling crosstalk, are significant in overcoming lethal breast cancer. The use of TGF-β inhibitors in combination with second-generation antiandrogen targeting AR (enzalutamide) is currently under intense exploration in other cancers. Recent studies revealed in preclinical models of advanced prostate cancer that the TGF-β receptor I inhibitor galunisertib, in combination with the AR-targeting antiandrogen enzalutamide, led to a significant reduction of tumor growth compared to the single treatments. Moreover, this treatment promoted the phenotypic reversion of EMT to MET and caused disturbance of the actin cytoskeleton remodeling (targeting cofilin), to deliver tumor suppression and therapeutic response (5). While the present study highlights the relationship between AR and TGF-β in anchorage independence in vitro, further studies are needed to elucidate the validity of using combined antiandrogen therapy and TGF-β inhibition using in vivo models. Thus, with TGF-β emerging as a critical navigator of the phenotypic transitions in highly aggressive breast tumors, other signaling effectors of this pathway regulating migration and focal adhesions must be also considered (talin, cofilin) (3). Transgenic mouse models such as the MMTV-PyMT provide a useful tool for examining the combined TGF-β/AR inhibition because these mice spontaneously develop mammary tumors that metastasize to the lungs and continue to express AR during tumor progression to late-stage lethal disease (8).

Unquestionably, this study enables a new understanding of the functional crosstalk between TGF-β and AR in conferring anoikis resistance, protecting the free cells from death, to drive progression of TNBC to lethal disease. The challenge is that TNBC remains an aggressive breast cancer subtype that claims the lives of thousands of women every year, with no targeted treatments currently in sight. The simultaneous pharmacological targeting platform to disarm TGF-β and AR from protecting the homeless cancer cells from dying (suggested by Rosas et al [2]), is impressive and calls for clinical trials toward improving the survival of patients with TNBC.