Teneale A. Stewart

Targeting EMT in cancer: opportunities for pharmacological intervention.

The spread of cancer cells to distant organs represents a major clinical challenge in the treatment of cancer. Epithelial-mesenchymal transition (EMT) has emerged as a key regulator of metastasis in some cancers by conferring an invasive phenotype. As well as facilitating metastasis, EMT is thought to generate cancer stem cells and contribute to therapy resistance. Therefore, the EMT pathway is of great therapeutic interest in the treatment of cancer and could be targeted either to prevent tumor dissemination in patients at high risk of developing metastatic lesions or to eradicate existing metastatic cancer cells in patients with more advanced disease. In this review, we discuss approaches for the design of EMT-based therapies in cancer, summarize evidence for some of the proposed EMT targets, and review the potential advantages and pitfalls of each approach.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1high/STIM2low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448–60. ©2011 AACR.

A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer.

Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells

Two-pore channel proteins, TPC1 and TPC2, are calcium permeable ion channels found localized to the membranes of endolysosomal calcium stores. There is increasing interest in the role of TPC-mediated intracellular signaling in various pathologies; however their role in breast cancer has not been extensively evaluated. TPC1 and TPC2 mRNA was present in all non-tumorigenic and tumorigenic breast cell lines assessed. Silencing of TPC2 but not TPC1 attenuated epidermal growth factor-induced vimentin expression in MDA-MB-468 breast cancer cells. This effect was not due to a general inhibition of epithelial to mesenchymal transition (EMT) as TPC2 silencing had no effect on epidermal growth factor (EGF)-induced changes on E-cadherin expression. TPC1 and TPC2 were also shown to differentially regulate cyclopiazonic acid (CPA)-mediated changes in cytosolic free Ca(2+). These findings indicate potential differential regulation of signaling processes by TPC1 and TPC2 in breast cancer cells.

Janus kinases and Src family kinases in the regulation of EGF-induced vimentin expression in MDA-MB-468 breast cancer cells

Epithelial-mesenchymal transition (EMT) is an important process associated with the metastasis of breast cancer cells. Members of the Janus kinases (JAKs) and Src family kinases (SFKs) are implicated in the regulation of an invasive phenotype in various cancer cell types. Using the pharmacological inhibitors JAK Inhibitor I (a pan-JAK inhibitor) and PP2 we investigated the role of the JAKs and SFKs, respectively, in the regulation of EMT markers in the MDA-MB-468 breast cancer cell line model of epidermal growth factor (EGF)-induced EMT. We identified selective inhibition of EGF induction of the mesenchymal marker vimentin by PP2 and JAK Inhibitor I. The effect of JAK Inhibitor I on vimentin protein induction occurred at a concentration lower than that required to significantly inhibit EGF-mediated signal transducer and activator of transcription 3 (STAT3)-phosphorylation, suggesting involvement of a STAT3-independent mechanism of EGF-induced vimentin regulation by JAKs. Despite our identification of a role for the JAK family in EGF-induced vimentin protein expression, siRNA-mediated silencing of each member of the JAK family was unable to phenocopy pharmacological inhibition, indicating potential redundancy among the JAK family members in this pathway. While SFKs and JAKs do not represent global regulators of the EMT phenotype, our findings have identified a role for members of these signaling pathways in the regulation of EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line.

Abstract P2-07-05: A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT), a process whereby tumorigenic epithelial cells acquire an invasive and migratory phenotype, is an important event in the invasion-metastasis cascade. As such, intracellular signaling pathways involved in the regulation of EMT represent potential therapeutic targets in the treatment and prevention of invasive cancer subtypes. The calcium ion, a highly versatile intracellular messenger, plays an important role in processes important in tumorigenesis including invasion and metastasis, and altered calcium signaling has been identified in various cancers. We recently identified that activation of signal transducer and activator of transcription 3 (STAT3) and expression of specific EMT markers in the MDA-MB-468 cell line model of epidermal growth factor (EGF) induced EMT display some calcium dependence. While the calcium permeable ion channel TRPM7 was shown to partially regulate this STAT3 activation and vimentin expression, the precise mechanisms of their regulation are not yet fully understood. The aim of this research was to investigate the upstream intracellular signaling pathway involved in EGF stimulated STAT3 activation and the subsequent induction of EMT in this model...

Calcium Channels and Pumps: Importance During Lactation as Potential Targets for Breast Cancer

The enrichment of milk with calcium is critical for the survival of mammals after birth. The process of transfer of calcium ions from the maternal blood supply into milk occurs through mammary alveolar epithelial cells. Recent research has provided deep mechanistic insight into these processes with candidates for the critical pathways involved in calcium transport identified. These proteins include the store-operated Ca2+ entry component Orai1 (basolateral Ca2+ influx), the secretory pathway Ca2+-ATPase isoform 2 (SPCA2, secretion of Ca2+), and the plasma membrane Ca2+-ATPase isoform 2 (PMCA2, apical membrane Ca2+ efflux). Increased expression of Orai1, SPCA2, and PMCA2 has also been identified in breast cancer cells; however, the remodeling of these targets often demonstrates selectivity for specific clinical and/or molecular subtypes. Silencing of these targets has identified roles for these proteins in the proliferation and/or migration of some breast cancer cell lines.

Abstract B57: Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts

Fibroblasts represent one of the predominant stromal cell populations present in the breast tumor microenvironment. Cancer-associated fibroblasts found growing in close proximity to the tumor site are thought to influence tumor behavior. While a number of protein and mRNA markers showing differential expression between cancer-associated and non-cancer associated fibroblasts have been proposed, current understanding of the phenotypic differences between these two cell populations remains incomplete. Calcium signaling plays an important role in each of the cancer hallmarks and is altered in a variety of disease states including cardiovascular disease. The aim of this study was to investigate the potential remodeling of the calcium signal and calcium channel and pump expression as a consequence of the transition from a normal to cancer-associated phenotype in human breast fibroblasts. To investigate expressional changes, RNA was isolated from primary cultures of matched normal and cancer-associated fibroblasts established from human breast cancer tissue specimens. Real time RT-PCR was used to assess mRNA levels of a total of 36 proteins involved in the movement of Ca2+ into and out of the cytoplasmic space, and included different classes of plasma membrane localized Ca2+ channels, as well as Ca2+ pumps of intracellular organelles. To further study potential differences in Ca2+ signaling between normal and cancer-associated fibroblast phenotypes, the immortalized human breast fibroblast line HMF3S was treated with transforming growth factor beta 1 (TGFβ1) (0, 0.01, 0.1, 1 and 10 ng/mL, 48 h), a growth factor implicated in the tumor microenvironment and myofibroblast differentiation. Successful TGFβ1-mediated activation of fibroblasts to a cancer-associated phenotype was confirmed by induction of smooth muscle alpha actin (SMαA) protein via immunoblotting. Real time RT-PCR was used to assess mRNA levels of classes of Ca2+ channels and pumps showing altered expression in patient matched normal and cancer-associated fibroblast pairs. In addition to assessing expression changes, potential functional differences in intracellular Ca2+ signaling profiles of HMF3S cells treated with TGFβ1 were assessed using a fluorometric imaging plate reader (FLIPRTETRA) and cells loaded with the Ca2+ sensitive indicator Fluo-4. Using both clinical samples, and an in vitro model of cancer-associated fibroblasts, our findings reveal a differential remodeling of plasma membrane calcium channels and organellar pumps of different classes in cells representative of cancer-associated relative to non-cancer associated fibroblasts. In addition to expression changes, there also appears to be a functional remodeling of the calcium signal in HMF3S fibroblasts induced to transition from a normal to cancer-associated phenotype by TGFβ1. This is the first study to assess changes in calcium pumps, channels and channel regulators, as well as remodeling of the calcium signal, in breast cancer-associated fibroblasts. This work may suggest that the breast cancer-associated fibroblast phenotype is associated with altered Ca2+ signaling. Citation Format: Teneale A. Stewart, Patsy S. Soon, Sarah J. Roberts-Thomson, Gregory R. Monteith. Assessment of gene expression of calcium pumps, channels, and channel regulators in human breast cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B57.