Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.
BACKGROUND
Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.


RESULTS
The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.


CONCLUSIONS
SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.
Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
Francesco
F
Lodola
Umberto
U
Laforenza
Elisa
E
Bonetti
Dmitry
D
Lim
Silvia
S
Dragoni
Cinzia
C
Bottino
Hwei Ling
HL
Ong
Germano
G
Guerra
Carlo
C
Ganini
Margherita
M
Massa
Mariangela
M
Manzoni
Indu S
IS
Ambudkar
Armando A
AA
Genazzani
Vittorio
V
Rosti
Paolo
P
Pedrazzoli
Franco
F
Tanzi
Francesco
F
Moccia
Camillo
C
Porta
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001896:Boron Compounds / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002292:Carcinoma, Renal Cell / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D052139:Intracellular Calcium-Sensing Proteins; D007680:Kidney Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009389:Neovascularization, Pathologic; D000071740:ORAI1 Protein; D061251:Primary Cell Culture; D015398:Signal Transduction / Q000187:drug effects; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism
D001896:Boron Compounds; D015220:Calcium Channels; D007211:Indoles; D052139:Intracellular Calcium-Sensing Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C572578:SARAF protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D002104:Cadmium; D007811:Lanthanum; D000255:Adenosine Triphosphate; C109986:2-aminoethoxydiphenyl borate; C000543:cyclopiazonic acid