Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.

Association of calcium sensing receptor polymorphisms at rs1801725 with circulating calcium in breast cancer patients.

BACKGROUND: Breast cancer (BC) patients with late-stage and/or rapidly growing tumors are prone to develop high serum calcium levels which have been shown to be associated with larger and aggressive breast tumors in post and premenopausal women respectively. Given the pivotal role of the calcium sensing receptor (CaSR) in calcium homeostasis, we evaluated whether polymorphisms of the CASR gene at rs1801725 and rs1801726 SNPs in exon 7, are associated with circulating calcium levels in African American and Caucasian control subjects and BC cases. METHODS: In this retrospective case-control study, we assessed the mean circulating calcium levels, the distribution of two inactivating CaSR SNPs at rs1801725 and rs1801726 in 199 cases and 384 age-matched controls, and used multivariable regression analysis to determine whether these SNPs are associated with circulating calcium in control subjects and BC cases. RESULTS: We found that the mean circulating calcium levels in African American subjects were higher than those in Caucasian subjects (p < 0.001). As expected, the mean calcium levels were higher in BC cases compared to control subjects (p < 0.001), but the calcium levels in BC patients were independent of race. We also show that in BC cases and control subjects, the major alleles at rs1801725 (G/T, A986S) and at rs1801726 (C/G, Q1011E) were common among Caucasians and African Americans respectively. Compared to the wild type alleles, polymorphisms at the rs1801725 SNP were associated with higher calcium levels (p = 0.006) while those at rs1801726 were not. Using multivariable linear mixed-effects models and adjusting for age and race, we show that circulating calcium levels in BC cases were associated with tumor grade (p = 0.009), clinical stage (p = 0.003) and more importantly, with inactivating mutations of the CASR at the rs1801725 SNP (p = 0.038). CONCLUSIONS: These data suggest that decreased sensitivity of the CaSR to calcium due to inactivating polymorphisms at rs1801725, may predispose up to 20% of BC cases to high circulating calcium-associated larger and/or aggressive breast tumors.

Identification of MAGEC2/CT10 as a High Calcium-Inducible Gene in Triple-Negative Breast Cancer.

The expression of the melanoma/cancer-testis antigen MAGEC2/CT10 is restricted to germline cells, but like most cancer-testis antigens, it is frequently upregulated in advanced breast tumors and other malignant tumors. However, the physiological cues that trigger the expression of this gene during malignancy remain unknown. Given that malignant breast cancer is often associated with skeletal metastasis and co-morbidities such as cancer-induced hypercalcemia, we evaluated the effect of high Ca2+ on the calcium-sensing receptor (CaSR) and potential mechanisms underlying the survival of triple-negative breast cancer (TNBC) cells at high Ca2+. We show that chronic exposure of TNBC cells to high Ca2+ decreased the sensitivity of CaSR to Ca2+ but stimulated tumor cell growth and migration. Furthermore, high extracellular Ca2+ also stimulated the expression of early response genes such as FOS/FOSB and a unique set of genes associated with malignant tumors, including MAGEC2. We further show that the MAGEC2 proximal promoter is Ca2+ inducible and that FOS/FOSB binds to this promoter in a Ca2+- dependent manner. Finally, downregulation of MAGEC2 strongly inhibited the growth of TNBC cells in vitro. These data suggest for the first time that MAGEC2 is a high Ca2+ inducible gene and that aberrant expression of MAGEC2 in malignant TNBC tissues is at least in part mediated by an increase in circulating Ca2+via the AP-1 transcription factor.

Diverse Roles of Annexin A6 in Triple-Negative Breast Cancer Diagnosis, Prognosis and EGFR-Targeted Therapies.

The calcium (Ca2+)-dependent membrane-binding Annexin A6 (AnxA6), is a multifunctional, predominantly intracellular scaffolding protein, now known to play relevant roles in different cancer types through diverse, often cell-type-specific mechanisms. AnxA6 is differentially expressed in various stages/subtypes of several cancers, and its expression in certain tumor cells is also induced by a variety of pharmacological drugs. Together with the secretion of AnxA6 as a component of extracellular vesicles, this suggests that AnxA6 mediates distinct tumor progression patterns via extracellular and/or intracellular activities. Although it lacks enzymatic activity, some of the AnxA6-mediated functions involving membrane, nucleotide and cholesterol binding as well as the scaffolding of specific proteins or multifactorial protein complexes, suggest its potential utility in the diagnosis, prognosis and therapeutic strategies for various cancers. In breast cancer, the low AnxA6 expression levels in the more aggressive basal-like triple-negative breast cancer (TNBC) subtype correlate with its tumor suppressor activity and the poor overall survival of basal-like TNBC patients. In this review, we highlight the potential tumor suppressor function of AnxA6 in TNBC progression and metastasis, the relevance of AnxA6 in the diagnosis and prognosis of several cancers and discuss the concept of therapy-induced expression of AnxA6 as a novel mechanism for acquired resistance of TNBC to tyrosine kinase inhibitors.

Reciprocal expression of Annexin A6 and RasGRF2 discriminates rapidly growing from invasive triple negative breast cancer subsets.

Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.

Implication of calcium activated RasGRF2 in Annexin A6-mediated breast tumor cell growth and motility.

The role of AnxA6 in breast cancer and in particular, the mechanisms underlying its contribution to tumor cell growth and/or motility remain poorly understood. In this study, we established the tumor suppressor function of AnxA6 in triple negative breast cancer (TNBC) cells by showing that loss of AnxA6 is associated with early onset and rapid growth of xenograft TNBC tumors in mice. We also identified the Ca2+ activated RasGRF2 as an effector of AnxA6 mediated TNBC cell growth and motility. Activation of Ca2+ mobilizing oncogenic receptors such as epidermal growth factor receptor (EGFR) in TNBC cells or pharmacological stimulation of Ca2+ influx led to activation, subsequent degradation and altered effector functions of RasGRF2. Inhibition of Ca2+ influx or overexpression of AnxA6 blocked the activation/degradation of RasGRF2. We also show that AnxA6 acts as a scaffold for RasGRF2 and Ras proteins and that its interaction with RasGRF2 is modulated by GTP and/or activation of Ras proteins. Meanwhile, down-regulation of RasGRF2 and treatment of cells with the EGFR-targeted tyrosine kinase inhibitor (TKI) lapatinib strongly attenuated the growth of otherwise EGFR-TKI resistant AnxA6 high TNBC cells. These data not only suggest that AnxA6 modulated Ca2+ influx and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2.