The role of calcium transporting proteins in the acquisition of resistance to trastuzumab

Abstract

HER2-positive breast cancers represent approximately 20-25% of all breast cancers and are characterized by an overexpression of the growth factor receptor HER2. Trastuzumab, a monoclonal antibody, is a molecularly targeted therapeutic used in the treatment of this subtype of breast cancer. However, 30% of eligible patients have intrinsic resistance to trastuzumab and approximately 60% of patients who initially responded to this therapeutic, develop resistance within one year. Calcium transporters and modulators are known to be involved in breast cancer and in chemoresistance. However, their role has not been evaluated in HER2-positive trastuzumab resistant breast cancer cells. The aim of this project was to identify possible calcium related proteins associated with trastuzumab resistance. In the first part of this thesis, the expression of Ca2+ transporters and modulators and their role in trastuzumab activity was assessed in the HER2-positive breast cancer cell line SKBR3. Ca2+ signaling profiling was also assessed using fluorescence imaging plate reader (FLIPR) assays. Inhibition of the expression of the Ca2+ channels TPC2, TRPV1 and the Ca2+ channel modulator STIM1 using siRNA decreased SKBR3 cellular proliferation. Silencing of STIM1, the Ca2+ pump SPCA1 and the Ca2+ permeable ion channel TRPM7 increased the anti-proliferative effects of trastuzumab in SKBR3 cells. In the second part of this thesis, trastuzumab resistant and age-matched control cell lines were established from parental SKBR3 cells through seven months of continuous culturing in the presence of trastuzumab. Two trastuzumab treated colonies were selected for their resistance to trastuzumab (RT1 and RT2). Two other colonies were selected from age-matched controls because of their development of de novo resistance to trastuzumab (RV1 and RV2). Two age-matched cell lines that retained their sensitivity to trastuzumab were selected as controls (SV1 and SV2). Levels of mRNA expression of 45 Ca2+ channels, pumps and channel modulators were evaluated using quantitative RT-PCR. An siRNA screen of selected targets to identify targets that when silenced could restore trastuzumab sensitivity was also performed. Additionally Ca2+ signaling profiling and the quantitation of HER2, EGFR and IGF1R protein expression were conducted. All trastuzumab resistant cell lines maintained their overexpression of the HER2 receptor. Significantly increased mRNA levels of the voltage-gated calcium Ca2+ channel CaV3.2 was observed in both de novo resistant cell lines RV1 and RV2 compared to control cell lines SV1 and SV2. Acquired resistant cell lines RT1 and RT2 showed altered sensitivity to the purinergic receptor activator ATP, indicating a possible remodeling of Ca2+ signaling in these trastuzumab resistant cell lines. In the third part of this thesis, specific experiments were conducted to further evaluate two selected targets, the Ca2+ permeable ion channels CaV3.2 and TRPM7 channel. Pharmacological inhibition and silencing of CaV3.2 channel did not reverse trastuzumab resistance. However, CaV3.2 mRNA levels were higher in the basal HER2-positive trastuzumab resistant HCC1569 breast cancer cell line compared to the luminal HER2-positive trastuzumab sensitive SKBR3 cell line. Partial siRNA-mediated silencing of TRPM7 or pharmacological inhibition of TRPM7 channel activity did not reverse trastuzumab resistance in the trastuzumab resistant cell line RV1. However, the TRPM7 kinase inhibitor NH125 was able to promote trastuzumab activity in the trastuzumab resistant cell line RV1. Further studies are required to definitively associate TRPM7 kinase with trastuzumab resistance, given the reported sensitivity of other atypical α-kinases to NH125. In the last part of this thesis publically available data was mined to identify other potential calcium related proteins associated with trastuzumab resistance. These data sets included cDNA microarray analysis of trastuzumab resistant and sensitive SKBR3 cell lines and trastuzumab resistant breast cancer clinical samples and proteomic analysis of trastuzumab resistant and sensitive SKBR3 cell lines. These analyses indicated that the Ca2+ ATPase pump SERCA3 and galectin-3 may be associated with trastuzumab resistance. Results presented in this thesis suggest that the acquisition of trastuzumab resistance may be associated with the expression and/or activity of specific Ca2+ channels and pumps, including SERCA3, CaV3.2 channel and TRPM7 and the Ca2+-related protein galectin-3. Further studies of these proteins may help identify new approaches to reverse trastuzumab resistance and/or identify new biomarkers for predicting trastuzumab sensitivity in HER2-positive breast cancers.