Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels’ Activation in Breast Cancer Cells

Abstract

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells’ malignant phenotype repression and reduction of their resistance to treatments.

Modulated TRPC1 Expression Predicts Sensitivity of Breast Cancer to Doxorubicin and Magnetic Field Therapy: Segue Towards a Precision Medicine Approach

“…Provocatively, these dichotomous proliferative and migratory responses to inflammatory conditions may represent a point of vulnerability to be exploited by PEMF-based therapies in order to mitigate the invasiveness of breast cancer cells. PEMF exposure attenuates the proliferative and migratory capacities of breast cancer cells in positive correlation to TRPC1 channel expression ( Figures 5 and 7 ), aligning with evidence that catalytic activation of TRPC6, similarly implicated with proliferation and inflammatory responses in breast cancer, attenuates MDA-MB-231 breast cancer cell viability and migratory capacity ( 51 ). Both studies further demonstrated reductions in breast cancer cell invasiveness in response to activation of either TRPC1 (PEMF exposure) ( Figure 7D ) or TRPC6 (Furin inhibition) ( 51 ).…”

“…PEMF exposure attenuates the proliferative and migratory capacities of breast cancer cells in positive correlation to TRPC1 channel expression ( Figures 5 and 7 ), aligning with evidence that catalytic activation of TRPC6, similarly implicated with proliferation and inflammatory responses in breast cancer, attenuates MDA-MB-231 breast cancer cell viability and migratory capacity ( 51 ). Both studies further demonstrated reductions in breast cancer cell invasiveness in response to activation of either TRPC1 (PEMF exposure) ( Figure 7D ) or TRPC6 (Furin inhibition) ( 51 ). Moreover, TRPC1 channel expression is greater in the highly metastatic MDA-MB-231 cell line compared to the less invasive MCF-7 cell line ( Figure 5E ) ( 52 ).…”

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells

“…SOCE has been reported to play an essential role in the development of a variety of breast cancer hallmarks, including cell viability, migration, proliferation, and apoptosis resistance [22][23][24][25]. STIM1 and Orai1 are the key elements in SOCE but several proteins, such as SARAF and EFHB, play an important role fine-tuning the amount of Ca 2+ entering the cell through the store-operated channels [13,14,16,17].…”

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors

“…Cells were routinely maintained in Dulbecco’s minimal essential medium (DMEM) (Gibco, Saint Aubin, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% sodium pyruvate, 0.1 mg/mL streptomycin, 100 U/mL penicillin at 37 °C in a 5% CO2-humidified atmosphere. For SW480, SW620, CT26, HT-29 tumorsphere formation mimicking CSCs phenotype, as previously described [ 32 ], cells were cultured in DMEM/F-12 supplemented with N2, B27 supplements (Thermo Fisher Scientific, Waltham, MA, USA), and growth factors [20 ng/mL basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF)] (Sigma, St. Louis, MO, USA) in a 96-well round bottom plate that facilitated the production of homogeneous spheroids of cells.…”

“…Previously, the inhibition of the convertases in CSCs was associated with upregulated expression of various metallothioneins known as tumor suppressor genes, of which the loss in colon cancer patients was associated with bad prognosis [ 31 ]. On the other hand, Furin was reported to interfere with calcium mobilization [ 29 , 32 ], and PCs repression reduced the malignant phenotype of cancer cells and resistance to apoptotic agents [ 29 ]. As a result, in recent years, the concept of CSCs has interesting implications in the exploration of new and effective therapies that target the altered signaling pathways of CSCs.…”