Regulates Breast Cancer Cell Migration Research Articles

Macrophage-secreted exosomes inhibit breast cancer cell migration via the miR- 101-3p/DLG5 axis

Objective: To investigate the role of macrophages in regulating breast cancer cell migration and its related mechanisms. Methods: Human leukemia monocytic cell line THP-1-secreted exosomes were isolated using multi-step ultracentrifugation and verified using nanoparticle tracking analysis. Differentially expressed miRNAs were identified using RNA sequencing. Overexpression of inhibitors of hsa-miR-101-3p in breast cancer MDA-MB-231 cells was performed by infecting their lentiviral constructs. The luciferase reporter assay was used to evaluate the interaction of DLG5 and miR-101. DGL5 expression was detected using qRT-PCR and Western blot analyses. Results: The migration of breast cancer cells was significantly inhibited after addition of exosomes. RNA sequencing results showed that miR-101-3p expression was significantly upregulated. Targetscan analysis predicted that miR-101-3p could target DLG5, and this prediction was verified using the luciferase assay. The addition of the miR-101-3p precursor significantly increased the expression of miR-101-3p, and the mRNA and protein levels of DLG5 were suppressed. In contrast, inhibiting the expression of miR-101-3p increased the mRNA and protein levels of DLG5. Furthermore, the scratch assay showed that inhibiting miR-101-3p could promote the migration of MDA-MB-231 cells. Conclusions: Macrophage exosomes can inhibit the migration of breast cancer cells, and increasing the expression of miR-101-3p to inhibit DLG5 expression may play an important role in this process, which needs further investigation.

Microrchidia 2/Histone Deacetylase 1 Complex Regulates E-cadherin Gene Expression and Function.

Although Microrchidia 2 (MORC2) is widely overexpressed in human malignancies and linked to cancer cell proliferation, metabolism, and metastasis, the mechanism of action of MORC2 in cancer cell migration and invasion is yet undeciphered. Here, we identified for the first time that MORC2, a chromatin remodeler, regulates E-cadherin expression and, subsequently regulates breast cancer cell migration and invasion. We observed a negative correlation between the expression levels of MORC2 and E-cadherin in breast cancer. Further, the overexpression of MORC2 resulted in decreased expression levels of E-cadherin. In addition, coimmunoprecipitation and chromatin immunoprecipitation assays revealed that MORC2 interacts with HDAC1 and gets recruited onto the E-cadherin promoter to inhibit its transcription, thereby suppress its expression. Consequently, knockdown of HDAC1 in MORC2-overexpressing cells led to reduced cancer cell migration and invasion. Interestingly, we noticed that MORC2 regulated glucose metabolism via c-Myc, and LDHA, also modulates the expression of E-cadherin. Collectively, these results demonstrate for the first time a mechanistic role for MORC2 as an upstream regulator of E-cadherin expression and its associated functions in breast cancer.

STIM1 signals through NFAT1 independently of Orai1 and SOCE to regulate breast cancer cell migration

Store-operated calcium entry (SOCE) contributes to several physiological and pathological conditions including transcription, secretion, immunodeficiencies, and cancer. SOCE has been shown to be important for breast cancer cell migration where knockdown of SOCE components (STIM1 or Orai1) decreases cancer metastasis. Here we show unexpectedly that complete knockout of STIM1 (STIM1-KO) using gene editing in metastatic MDA-MB-231 breast cancer cells results in faster migration and enhanced invasion capacity. In contrast, Orai1-KO cells, which have similar levels of SOCE inhibition as STIM1-KO, migrate slower than the parental cell line. This shows that the enhanced migration phenotype of STIM1-KO cells is not due to the loss of Ca2+ entry through SOCE, rather it involves transcriptional remodeling as elucidated by RNA-seq analyses. Interestingly, NFAT1 is significantly downregulated in STIM1-KO cells and overexpression of NFAT1 reversed the enhanced migration of STIM1-KO cells. STIM1 knockout in other breast cancer cells, independent of their metastatic potential, also enhanced cell migration while reducing NFAT1 expression. These data argue that in breast cancer cells STIM1 modulates NFAT1 expression and cell migration independently of its role in SOCE.

TMBIM6-mediated miR-181a expression regulates breast cancer cell migration and invasion via the MAPK/ERK signaling pathway.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) has been reported to regulate cell death pathways and is overexpressed in several types of cancers. In this study, we investigated whether high expression of TMBIM6 in breast cancer was significantly associated with cancer invasiveness. Knockdown of TMBIM6 reduced proliferation and migration of invasive breast cancer cells through downregulation of the MAPK/ERK signaling pathway. Moreover, we suggested that expression of miR-181a was significantly suppressed upon TMBIM6 knockdown. In contrast, overexpression of TMBIM6 significantly increased cell invasion and migration through up-regulation of mesenchymal markers and matrix metalloproteinase-9 (MMP-9) and enhanced activation of the MAPK/ERK signaling pathway. We also observed that up-regulation of TMBIM6 significantly increased the expression of miR-181a by TMBIM6-mediated pathway. TMBIM6 and miR-181a-mediated ERK activation induced the expression of Snail-1 and Snail-2 in FOSL-1/C-JUN-dependent manner. Overall, our data demonstrated that TMBIM6-induced miR-181a up-regulation plays an important role in the efficient modulation of migration and invasion of breast cancer cells.

C/EBP\u03b2 isoform-specific regulation of migration and invasion in triple-negative breast cancer cells

The transcription factor C/EBPβ is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPβ-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPβ-isoforms in breast cancer development may reveal new avenues of treatment.

Orai3 Calcium Channel Regulates Breast Cancer Cell Migration through Calcium-Dependent and -Independent Mechanisms.

Orai3 calcium (Ca2+) channels are implicated in multiple breast cancer processes, such as proliferation and survival as well as resistance to chemotherapy. However, their involvement in the breast cancer cell migration processes remains vague. In the present study, we exploited MDA-MB-231 and MDA-MB-231 BrM2 basal-like estrogen receptor-negative (ER−) cell lines to assess the direct role of Orai3 in cell migration. We showed that Orai3 regulates MDA-MB-231 and MDA-MB-231 BrM2 cell migration in two distinct ways. First, we showed that Orai3 remodels cell adhesive capacities by modulating the intracellular Ca2+ concentration. Orai3 silencing (siOrai3) decreased calpain activity, cell adhesion and migration in a Ca2+-dependent manner. In addition, Orai3 interacts with focal adhesion kinase (FAK) and regulates the actin cytoskeleton, in a Ca2+-independent way. Thus, siOrai3 modulates cell morphology by altering F-actin polymerization via a loss of interaction between Orai3 and FAK. To summarize, we demonstrated that Orai3 regulates cell migration through a Ca2+-dependent modulation of calpain activity and, in a Ca2+-independent manner, the actin cytoskeleton architecture via FAK.

Auto-inhibitory intramolecular S5/S6 interaction in the TRPV6 channel regulates breast cancer cell migration and invasion

TRPV6, a Ca-selective channel, is abundantly expressed in the placenta, intestine, kidney and bone marrow. TRPV6 is vital to Ca homeostasis and its defective expression or function is linked to transient neonatal hyperparathyroidism, Lowe syndrome/Dent disease, renal stone, osteoporosis and cancers. The fact that the molecular mechanism underlying the function and regulation of TRPV6 is still not well understood hampers, in particular, the understanding of how TRPV6 contributes to breast cancer development. By electrophysiology and Ca imaging in Xenopus oocytes and cancer cells, molecular biology and numerical simulation, here we reveal an intramolecular S5/S6 helix interaction in TRPV6 that is functionally autoinhibitory and is mediated by the R532:D620 bonding. Predicted pathogenic mutation R532Q within S5 disrupts the S5/S6 interaction leading to gain-of-function of the channel, which promotes breast cancer cell progression through strengthening of the TRPV6/PI3K interaction, activation of a PI3K/Akt/GSK-3β cascade, and up-regulation of epithelial-mesenchymal transition and anti-apoptosis.

Cell surface GRP78 and Dermcidin cooperate to regulate breast cancer cell migration through Wnt signaling.

The heat shock protein GRP78 typically resides in the endoplasmic reticulum in normal tissues, but it has been shown to be expressed on the cell surface of several cancer cells, and some stem cells, where it can act as a signaling molecule by not-yet-fully defined mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that sGRP78 can function in a cell-context dependent manner, with a diverse array of reported binding partners, to regulate a variety of cellular responses. We had previously shown that sGRP78 was important in regulating pluripotent stem cell (PSC) functions, and hypothesized that embryonic-like mechanisms of GRP78 were critical to regulating aggressive breast cancer cell functions. Here, using proteomics we identify Dermcidin (DCD) as a novel sGRP78 binding partner common to both PSCs and breast cancer cells. We show that GRP78 and DCD cooperate to regulate stem cell and cancer cell migration that is dependent on the cell surface functions of these proteins. Finally, we identify Wnt/β-catenin signaling, a critical pathway in stem cell and cancer cell biology, as an important downstream intermediate in regulating this migration phenotype.

Annilin regulates breast cancer cell migration, growth and metastasis by non‐canonical mechanisms involving control of cell stemness and differentiation.

Breast cancer metastasis is driven by profound remodeling of the intracellular cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules and septin polymers. It is markedly overexpressed in breast cancer and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer metastasis. CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA‐MB‐231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by a combination of wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesions was evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar grow, whereas cancer stem cells were examined using a mammoshpere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo was determined by injecting control and anillin‐depleted breast cancer cells into NGS mice. The loss‐of‐function and gain‐of‐function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion and anchorage‐independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus, however knockout of this protein affected the cytoplasmic/cortical events, such as the organization of actin cytoskeleton and cell‐matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin‐depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans‐differentiation. Such trans‐differentiation was manifested by upregulation of basal keratins along with increased expression of E‐cadherin and P‐cadherin. Knockdown of E‐cadherin reversed attenuated migration and invasion of anillin‐deficient breast cancer cells. Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

Circular RNA CircVAPA Regulates Breast Cancer Cell Migration and Invasion Via Sponging miR-130a-5p

Aim: We aimed to explore the roles of circular RNA, circVAPA in regulating cell migration and invasion of breast cancer. Materials & methods: CircVAPA expression was detected in breast cancer tissues and cells. The role of circVAPA was evaluated by MTT assay, wound-healing and transwell assay. The relationship between circVAPA and miR-130a-5p and the location of circVAPA were explored. Results: We discovered that circVAPA was dysregulated in breast cancer tissues and cells. Ectopic circVAPA regulated breast cancer migration, invasion and proliferation. CircVAPA was mainly expressed in the cytoplasm and could act as a miRNA sponge for miR-130a-5p, but did not regulate its parental gene. Conclusion: CircVAPA may promote migration and invasion capacity of breast cancer via harboring miR-130a-5p.

Anillin regulates breast cancer cell migration, growth, and metastasis by non-canonical mechanisms involving control of cell stemness and differentiation

BackgroundBreast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis.MethodsCRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice.ResultsLoss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells.ConclusionOur study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

Arl13b Regulates Breast Cancer Cell Migration and Invasion by Controlling Integrin-Mediated Signaling.

Breast cancer is the first cause of cancer-related mortality among women worldwide, according to the most recent estimates. This mortality is mainly caused by the tumors’ ability to form metastases. Cancer cell migration and invasion are essential for metastasis and rely on the interplay between actin cytoskeleton remodeling and cell adhesion. Therefore, understanding the mechanisms by which cancer cell invasion is controlled may provide new strategies to impair cancer progression. We investigated the role of the ADP-ribosylation factor (Arf)-like (Arl) protein Arl13b in breast cancer cell migration and invasion in vitro, using breast cancer cell lines and in vivo, using mouse orthotopic models. We show that Arl13b silencing inhibits breast cancer cell migration and invasion in vitro, as well as cancer progression in vivo. We also observed that Arl13b is upregulated in breast cancer cell lines and patient tissue samples. Moreover, we found that Arl13b localizes to focal adhesions (FAs) and interacts with β3-integrin. Upon Arl13b silencing, β3-integrin cell surface levels and FA size are increased and integrin-mediated signaling is inhibited. Therefore, we uncover a role for Arl13b in breast cancer cell migration and invasion and provide a new mechanism for how ARL13B can function as an oncogene, through the modulation of integrin-mediated signaling.

Brahma related gene 1 (BRG1) regulates breast cancer cell migration and invasion by activating MUC1 transcription

Mucin 1 (MUC1) is a versatile oncoprotein that promotes the migration and invasion of breast cancer cells. MUC1 is up-regulated by pro-metastatic stimuli in breast cancer cells. The underlying epigenetic mechanism, however, is not fully understood. Here we report that brahma related gene 1 (BRG1), a chromatin remodeling protein, was up-regulated in highly malignant breast cancer biopsy specimens compared to those of lesser malignancy. Over-expression of BRG1 potentiated whereas depletion of BRG1 attenuated migration and invasion of MCF-7 cells. BRG1 directly bound to the MUC1 promoter and activated MUC1 transcription in a STAT1-and RelA-dependent manner. BRG1 contributed to MUC1 trans-activation by modulating histone modifications surrounding the MUC1 promoter. Specifically, BRG1 interacted with and recruited KDM3A to demethylate H3K9 on the MUC1 promoter thereby activating MUC1 transcription. Depletion of KDM3A ameliorated migration and invasion of MCF-7 cells. Altogether, our data elucidate a novel epigenetic pathway that links MUC1 transcription to breast cancer metastasis.

Talin2 regulates breast cancer cell migration and invasion by apoptosis.

Talin is a key component molecule of the extracellular matrix-integrin-cytoskeleton. It serves an important role in the activation of integrin, which, in turn, is known to mediate physiological and pathological processes, including cell adhesion, growth, tumorigenesis, and metastasis. In vertebrates, there are two Talin genes, Talin1 and Talin2. Talin1 is known to regulate focal adhesion dynamics, cell migration and cell invasion; however, the precise role of Talin2 in cancer remains unclear. In the present study, the functional role of Talin2 was examined in the MDA-MB-231 breast cancer cell line. Talin2 knockdown significantly inhibited growth, migratory capacity and invasiveness of MDA-MB-231 cells, and promoted apoptosis. The expression levels of Talin2 in breast cancer cells and in the peritumoral normal breast tissues were also determined by immunohistochemistry. Talin2 was identified to be overexpressed in breast cancer tissues compared with that in the peritumoral breast tissues. In addition, the knockdown of Talin2 by specific RNA interference markedly inhibited cell growth, and caused the downregulation of the apoptotic markers, cleaved Caspase-3 and phosphorylation of poly ADP-ribose polymerase. These findings demonstrate that Talin2 expression is upregulated in human breast cancer and that downregulation of Talin2 may serve as a useful therapeutic target in patients with breast cancer.

Differential prognostic impact of interleukin-34 mRNA expression and infiltrating immune cell composition in intrinsic breast cancer subtypes.

Interleukin-34 (IL-34) is a ligand for the CSF-1R and has also two additional receptors, PTPRZ1 and syndecan-1. IL-34 plays a role in innate immunity, inflammation, and cancer. However, the role of IL-34 in breast cancer is still ill-defined. We analyzed IL-34 mRNA expression in breast cancer cell lines and breast cancer patients and applied established computational approaches (CIBERSORT, ESTIMATE, TIMER, TCIA), to analyze gene expression data from The Cancer Genome Atlas (TCGA). Expression of IL-34 was associated with a favorable prognosis in luminal and HER2 but not basal breast cancer patients. Gene expression of CSF-1 and CSF-1R was strongly associated with myeloid cell infiltration, while we found no or only weak correlations between IL-34, PTPRZ1, syndecan-1 and myeloid cells. In vitro experiments showed that tyrosine phosphorylation of CSF-1R, ERK, and FAK and cell migration are differentially regulated by IL-34 and CSF-1 in breast cancer cell lines. Collectively, our data suggest that correlation of IL-34 gene expression with survival is dependent on the molecular breast cancer subtype. Furthermore, IL-34 is not associated with myeloid cell infiltration and directly regulates breast cancer cell migration and signaling.

MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1− MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

Autoubiquitination of feline E3 ubiquitin ligase BCA2

BCA2/RNF115/Rabring7 is a RING type E3 ubiquitin ligase that is overexpressed in human breast tumors and is important for regulating breast cancer cell migration. In the present investigation, feline BCA2 (fBCA2) was identified and characterized. Compared with its human counterpart, the fBCA2 cDNA was confirmed to be 918 base pairs in length showing 92.6% consensus and identity positions, encoding a protein of 305 amino acids with 96.7% consensus and 93.1% identity positions. The fBCA2 protein contains a RING domain at the C-terminus, which was found to be essential for its autoubiquitination.

MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. Our previous study has shown that MRTF-A promote the migration of MDA-MB-231 cells and WDR1 promotes breast cancer cell migration. But the exact molecular mechanism on metastasis is still not fully understood, we now report that WDR1 enhanced the effect of MRTF-A induced-MDA-MB-231 cell migration by promoting the expression of the EMT markers and migration markers via RhoA-MRTF-A signaling pathway. Importantly, WDR1 promoted the nuclear importion of MRTF-A by affecting the expression of nuclear transport protein importin. But WDR1 did not affect the expression of MRTF-A. Interestingly, MRTF-A promoted the expression of miR-206 via its promoter CArG box but miR-206 inhibits the migration of breast cancer cells through suppressing the expression of WDR1 and MRTF-A via targeted their 3′UTR. Our data thus provide important and novel insights into MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration.

Abstract 890: The role of calpastatin isoforms in breast cancer progression

Abstract Calpastatin is the endogenous inhibitor of the ubiquitously expressed m-calpain and μ-calpain. Both of them are important regulators of various cellular pathways including proliferation, apoptosis, adhesion, and migration. Dysregulation of the calpain system is associated with a wide range of pathologies including tumour invasion and metastasis. In breast cancer, down-regulation of calpastatin mRNA, in particular an exon 3 containing variant, is associated with lymphatic vessel invasion which is the predominant form of lymphovascular invasion (LVI) (96%), a critical initial step of metastases. The current project seeks to determine the role that individual calpastatin isoforms play in regulating breast cancer cell migration and endothelial interactions to understand processes involved in regulating LVI. Seven breast cancer cell lines were assessed for calpain system protein expression (calpain-1, -2, -4 and calpastatin) and calpastatin (CAST) mRNA expression (total CAST, CASTexon3, CAST I, II and III) by Western blotting and qRT-PCR, respectively. T47D and MDA-MB-231 cell lines showed low calpastatin but high calpain expression. From haptotaxis cell migration data, T47D migrated more slowly than MDA-MB-231 (35.49±6.14 and 80.34±5.52 percent wound closure at 24 hours post-scratch respectively) (P value=0.0233). These cell lines, that represent different subtypes of breast cancer (luminal and basal respectively), were therefore selected to study the role of differential calpastatin type/isoform expression. GFP-tagged XL and Leader domains of calpastatin type I, II and III, with or without exon 3 were overexpressed in each cell line. The different calpastatin types show differential subcellular localization. In MDA-MB-231, type I, III, IΔ3, IIIΔ3 were expressed generally in cytoplasm whereas type II and IIΔ3 were located as punctate nuclear invaginations. To assess if overexpression of the different calpastatin types cause phenotypic changes, full length HA-tagged calpastatin type I, II and III were transfected into MDA-MB-231 and T47D and stable clones produced. Thus far stable type II overexpression in MDA-MB-231 and type III in T47D have been obtained with others under single cell selection. The localization of full length type II in MDA-MB-231 was similar to the localization of the truncated form that showing a strong single perinuclear signal. In T47D, the full length and truncated type III also show similarly localization that expressed throughout the cytoplasm. For the effect of calpastatin types on cell proliferation, calpastatin type II in MDA-MB-231, and type III in T47D, had no significant effect on cell doubling time when compared to respective controls (P value=0.6400 and 0.8874, respectively). According to preliminary studies, calpastatin type II seems to have no effect on clonogenicity of MDA-MB-231. Such effect on T47D and other phenotypic changes involved in regulating LVI of transfected cell are being examined. Citation Format: Bhudsaban Sukkarn, Sarah Storr, Ian O. Ellis, Kirsty Jewell, Tim Parr, Stewart G. Martin. The role of calpastatin isoforms in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 890. doi:10.1158/1538-7445.AM2017-890

TRPM7 kinase domain rather than the channel regulates breast cancer cell migration and tumor metastasis

The channel‐kinase TRPM7 (transient receptor potential melastatin 7) is a bifunctional protein consisting of a cation channel that is permeable to Mg2+, Ca2+ and Mn2+ ions fused to a C‐terminal kinase domain. A growing number of studies with clinical significance suggest that TRPM7 is linked to adhesion and migration of breast cancer cells and promotes breast tumor metastasis. While the channel properties of TRPM7 have been studied extensively, little is known about its kinase activity and how it is linked to the control of actomyosin contractility. To understand the functions of the kinase domain we identified the first cell‐permeable inhibitor of the kinase domain (TRPM7‐IN‐1) and developed MDA‐MB‐231 cell lines in which TRPM7 is knocked out by CRISPR/Cas9 (KO), and in which various forms of TRPM7 were stably re‐expressed. These were wild type TRPM7 (WT), a kinase‐inactive mutant of TRPM7 (KD), and TRPM7 containing a truncated kinase domain (KT). TRPM7 KO significantly inhibited MDA‐MB‐231 cells migration. Only expression of the wild type TRPM7 (WT) rescued the migration phenotype, supporting a role for the kinase domain and not the channel in the regulation of cell migration. TRPM7‐IN‐1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA‐MB‐231 cells. And when MDA‐MB‐231 cells were treated with increasing doses of TRPM7‐IN‐1, TRPM7 phosphorylation at Ser‐1569 and its downstream Myosin IIa phosphorylation at Ser1943 were completely abrogated at a concentration of 5 μM. TRPM7‐IN‐1 inhibited MDA‐MB‐231 and BT 549 cell migration and invasion, while treatment of the KO cells with TRPM7‐IN‐1 showed no further inhibition of migration. Electrophysiological assessment of the TRPM7 channel revealed that the inhibitor did not affect the channel function in MD‐MB‐231 cells, supporting the notion that the inhibitor affects the migration exclusively through the inhibition of the TRPM7 kinase domain. Finally, in an experimental metastasis model, TRPM7‐IN‐1 significantly impeded metastasis to the lung. In conclusion, inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis.Support or Funding InformationFinancial support by grants from the National Institute of General Medical Sciences (R01GM059802), the Welch Foundation (F‐1390) and Cancer Prevention and Research Institute of Texas to KND.