Kunsala T. D. S. Yapa

TRPC1 is a differential regulator of hypoxia-mediated events and Akt signalling in PTEN-deficient breast cancer cells

ABSTRACT Hypoxia is a feature of the tumour microenvironment that promotes invasiveness, resistance to chemotherapeutics and cell survival. Our studies identify the transient receptor potential canonical-1 (TRPC1) ion channel as a key component of responses to hypoxia in breast cancer cells. This regulation includes control of specific epithelial to mesenchymal transition (EMT) events and hypoxia-mediated activation of signalling pathways such as activation of the EGFR, STAT3 and the autophagy marker LC3B, through hypoxia-inducible factor-1α (HIF1α)-dependent and -independent mechanisms. TRPC1 regulated HIF1α levels in PTEN-deficient MDA-MB-468 and HCC1569 breast cancer cell lines. This regulation arises from effects on the constitutive translation of HIF1α under normoxic conditions via an Akt-dependent pathway. In further support of the role of TRPC1 in EMT, its expression is closely associated with EMT- and metastasis-related genes in breast tumours, and is enhanced in basal B breast cancer cell lines. TRPC1 expression is also significantly prognostic for basal breast cancers, particularly those classified as lymph node positive. The defined roles of TRPC1 identified here could be therapeutically exploited for the control of oncogenic pathways in breast cancer cells. Summary: TRPC1 Ca2+ channels mediate hypoxia-associated cellular events via hypoxia-inducible factor 1-alpha (HIF1α)-dependent and -independent pathways in PTEN-deficient breast cancer cells.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

The voltage gated Ca2+-channel Cav3.2 and therapeutic responses in breast cancer

BackgroundUnderstanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca2+)-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca2+-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca2+-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome.MethodsExpression levels of a panel of Ca2+-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort).ResultsIncreased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers.ConclusionOur study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca2+-signalling in breast cancer progression and treatment.

Assessment of the TRPM8 inhibitor AMTB in breast cancer cells and its identification as an inhibitor of voltage gated sodium channels

Aims To assess levels of the calcium permeable transient receptor potential cation channel, subfamily melastatin, member 8 (TRPM8) in breast cancer molecular subtypes and to assess the consequences of TRPM8 pharmacological inhibition with AMTB (an inhibitor of TRPM8) on breast cancer cell lines. Materials and methods Cell viability and migration of breast cancer cells was determined using MTS assays and wound healing assays, respectively. RNA‐Seq analysis of breast tumours and qPCR in breast cancer cell lines were used to assess mRNA levels of ion channels. Membrane potential assays were employed to assess the effects of AMTB against specific voltage gated sodium channels (NaV). Key findings TRPM8 levels were significantly higher in breast cancers of the basal molecular subtype. AMTB decreased viable cell number in MDA‐MB‐231 and SK‐BR‐3 breast cancer cell lines (30 and 100 &mgr;M), and also reduced the migration of MDA‐MB‐231 cells (30 &mgr;M). However, these effects were independent of TRPM8, as no TRPM8 mRNA was detected in MDA‐MB‐231 cells. AMTB was identified as an inhibitor of NaV isoforms. NaV1.1–1.9 were expressed in a number of breast cancer cell lines, with NaV1.5 mRNA highest in MDA‐MB‐231 cells compared to the other breast cancer cell lines assessed. Significance TRPM8 levels may be elevated in basal breast cancers, however, TRPM8 expression appears to be lost in many breast cancer cell lines. Some of the effects of AMTB attributed to TRPM8 may be due to effects on NaV channels.