Carmine De Angelis

Molecular Mechanism and Clinical Implications of Endocrine Therapy Resistance in Breast Cancer

Endocrine therapy, the first targeted therapy in oncology, is the most successful systemic therapy in the management of estrogen receptor (ER)-positive breast cancer. Approximately 50% of patients with advanced disease do not respond to first-line treatment with tamoxifen, and many women who receive tamoxifen as adjuvant therapy experience tumor relapse and die from their disease. Aromatase inhibitors are proving superior to tamoxifen, at least in certain patient subsets. However, the response rate to these compounds is only slightly higher than that to tamoxifen in patients with advanced breast cancer, and both de novo or acquired resistance also occur, limiting the efficacy of the treatment. Advanced studies of ER biology have highlighted the role of an intimate cross talk between the ER and HER2/growth factor signaling pathways as a fundamental contributor to the development of resistance to hormone therapies. The aim of this review article is to summarize the current knowledge on mechanisms of resistance of breast cancer cells to endocrine therapies due to the cross talk between the ER and growth factor signaling pathways, and to explore newly available therapeutic strategies that could prolong duration of response and circumvent endocrine-resistant tumor growth.

The changing role of ER in endocrine resistance.

Estrogen receptor (ER) is expressed in approximately 70% of newly diagnosed breast tumors. Although endocrine therapy targeting ER is highly effective, intrinsic or acquired resistance is common, significantly jeopardizing treatment outcomes and minimizing overall survival. Even in the presence of endocrine resistance, a continued role of ER signaling is suggested by several lines of clinical and preclinical evidence. Indeed, inhibition or down-regulation of ER reduces tumor growth in preclinical models of acquired endocrine resistance, and many patients with recurrent ER+ breast tumors progressing on one type of ER-targeted treatment still benefit from sequential endocrine treatments that target ER by a different mechanism. New insights into the nature and biology of ER have revealed several mechanisms sustaining altered ER signaling in endocrine-resistant tumors, including deregulated growth factor receptor signaling that results in ligand-independent ER activation, unbalanced ER co-regulator activity, and genomic alterations involving the ER gene ESR1. Therefore, biopsies of recurrent lesions are needed to assess the changes in epi/genomics and signaling landscape of ER and associated pathways in order to tailor therapies to effectively overcome endocrine resistance. In addition, more completely abolishing the levels and activity of ER and its co-activators, in combination with selected signal transduction inhibitors or agents blocking the upstream or downstream targets of the ER pathway, may provide a better therapeutic strategy in combating endocrine resistance.

Overcoming Treatment Resistance in HER2-Positive Breast Cancer

Human epidermal growth factor receptor (HER)-2 overexpression or amplification occurs in about 20% of all breast cancers and results in a worse prognosis. Nevertheless, anti-HER2 treatments have recently been developed, resulting in dramatic improvements in the clinical outcome of patients with HER2-positive breast cancer. Trastuzumab has shown efficacy in early and advanced breast cancer treatment and lapatinib is currently approved for the treatment of advanced disease. Other anti-HER2 agents are being investigated. Mechanisms of resistance to trastuzumab treatment include crosstalk with heterologous receptors and amplification of HER2 signalling; amplification of the phosphoinositide 3-kinase (PI3K)/AKT pathway; alteration in binding of trastuzumab to HER2; and loss of HER2 expression. Proposed mechanisms of resistance to lapatinib involve derepression and/or activation of compensatory survival pathways through increased PI3K/AKT or estrogen receptor (ER) signalling. Several strategies to overcome resistance to anti-HER2 treatment are in different phases of development and include treatment with pertuzumab, T-DM1 and mammalian target of rapamycin (mTOR) inhibitors.

Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

Purpose: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples. Experimental design: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro. Results: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER−) converted to ER+ upon anti-HER2 therapy. In ER−/HER2+ MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti–HER2-resistant growth (P < 0.0001). Conclusions: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance. Clin Cancer Res; 21(17); 3995–4003. ©2015 AACR.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer

Significance One of the mechanisms of endocrine resistance in estrogen receptor α (ER)-positive (+) breast cancer is the cross-talk between the ER and growth factor receptor pathways leading to altered ER activity and a reprogrammed ER-dependent transcriptome. However, key mediators of this ER-dependent transcriptional reprogramming remain elusive. Here we demonstrate that forkhead box protein A1 (FOXA1) up-regulation via gene amplification or overexpression contributes to endocrine resistance and increased invasiveness phenotypes by altering the ER-dependent transcriptome. We further show that IL-8, one of the top altered FOXA1/ER effectors, plays a key role in mediating these phenotypes and is a potential target to treat ER+/FOXA1-high breast cancer. Our findings provoke a new interplay of FOXA1 in the ER transcriptional program in endocrine-resistant breast cancer. Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance

Significance Resistance to endocrine treatment remains a significant clinical obstacle. ESR1 mutations were found to be the mechanism of endocrine resistance in a substantial number of patients with metastatic ER-positive breast. However, these mutations are primarily linked to aromatase inhibitor resistance and are not strongly associated with tamoxifen resistance. Herein, we show that tamoxifen treatment promotes a RUNX2–ER complex, which mediates an altered ER cistrome that facilitates the up-regulation of SOX9. We show that up-regulation of SOX9, an embryonic transcription factor with key roles in metastases, is a driver of endocrine resistance in the setting of tamoxifen treatment. Our data provide putative targets for the development of new strategies to treat tamoxifen-resistant breast cancer. The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2–ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

The Evolving Role of the Estrogen Receptor Mutations in Endocrine Therapy-Resistant Breast Cancer

Recurrent ligand-binding domain ESR1 mutations have recently been detected in a substantial number of patients with metastatic ER+ breast cancer and evolve under the selective pressure of endocrine treatments. In this review, we evaluate the current understanding of the biological and clinical significance of these mutations. The preclinical studies revealed that these mutations lead to constitutive ligand-independent activity, indicating resistance to aromatase inhibitors and decreased sensitivity to tamoxifen and fulvestrant. Retrospective analyses of ESR1 mutations in baseline plasma circulating tumor DNA from completed clinical trials suggest that these mutations are prognostic and predictive of resistance to aromatase inhibitors in metastatic disease. Currently, we are lacking prospective studies to confirm these results and to determine the optimal treatment combinations for patients with the ESR1 mutations. In addition, the clinical development of novel agents to overcome resistance engendered by these mutations is also needed.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Blockade of AP-1 Potentiates Endocrine Therapy and Overcomes Resistance

The transcription factor AP-1 is downstream of growth factor (GF) receptors (GFRs) and stress-related kinases, both of which are implicated in breast cancer endocrine resistance. Previously, we have suggested that acquired endocrine resistance is associated with increased activity of AP-1 in an in vivo model. In this report, we provide direct evidence for the role of AP-1 in endocrine resistance. First, significant overlap was found between genes modulated in tamoxifen resistance and a gene signature associated with GF-induced estrogen receptor (ER) cistrome. Interestingly, these overlapping genes were enriched for key signaling components of GFRs and stress-related kinases and had AP-1 motifs in their promoters/enhancers. Second, to determine a more definitive role of AP-1 in endocrine resistance, AP-1 was inhibited using an inducible dominant-negative (DN) cJun expressed in MCF7 breast cancer cells in vitro and in vivo. AP-1 blockade enhanced the antiproliferative effect of endocrine treatments in vitro, accelerated xenograft tumor response to tamoxifen and estrogen deprivation in vivo, promoted complete regression of tumors, and delayed the onset of tamoxifen resistance. Induction of DN-cJun after the development of tamoxifen resistance resulted in dramatic tumor shrinkage, accompanied by reduced proliferation and increased apoptosis. These data suggest that AP-1 is a key determinant of endocrine resistance by mediating a global shift in the ER transcriptional program. Implications: AP-1 represents a viable therapeutic target to overcome endocrine resistance. Mol Cancer Res; 14(5); 470–81. ©2016 AACR.

De-escalation of treatment in HER2-positive breast cancer: Determinants of response and mechanisms of resistance

Overexpression and/or gene amplification of HER2, a crucial member of the HER family of four receptors, occur in about 15-20% of breast cancers and define an aggressive subtype of the disease. Activated HER homo and heterodimers govern a complex and redundant downstream signaling network that regulates cell survival and metastasis. Despite treatment with effective HER2-targeted therapies, many HER2-positive tumors fail to respond, or initially respond but eventually develop resistance. One of the upfront reasons for this treatment failure is failure to accurately select the tumors that are truly dependent on HER2 for survival and so would benefit the most from HER2-targeted therapy. In these truly HER2-addicted tumors (i.e. physiologically dependent), resistance could be the result of an incomplete inhibition of signaling at the HER receptor layer. In this regard, preclinical and clinical studies have documented the superiority of combination anti-HER2 therapy over single agent therapy to achieve a more comprehensive inhibition of the various HER receptor dimers. HER2 can be further activated or reactivated by mutations or other alterations in HER2 itself, or in other HER family members. Even when a complete and sustained HER inhibition is achieved, resistance to anti-HER therapy can arise by other somewhat dominant mechanisms, including preexisting or emerging alternative signaling pathways such as the estrogen receptor, deregulated downstream signaling components, especially of the PI3K pathway, and the tumor immune microenvironment. Most of the clinical trials that have investigated the efficacy of anti-HER2 therapies took place in the background of aggressive chemotherapy regimens, thus confounding the identification of key factors of resistance to the anti-HER2 treatments. Recent studies, however, have suggested that some HER2-amplified tumors may benefit from anti-HER2 therapy combined with only a single chemotherapy agent or in the absence of any chemotherapy. This de-escalation approach, a promising therapeutic strategy, is currently being explored in the clinic. In this review, we summarize the major molecular determinants that play a crucial role in influencing tumor response and resistance to HER2-targeted therapy, and discuss the growing need for patient stratification in order to facilitate the development of de-escalation strategies using HER2-targeted therapy alone with no chemotherapy.