A Truncated‐Flt1 Isoform of Breast Cancer Cells Is Upregulated by Notch and Downregulated by Retinoic Acid

Abstract

We have previously reported that the major isoform of Flt1/VEGFR-1 expressed in MDA-MB-231 breast cancer cells was a truncated intracellular isoform transcribed from intron 21 (i21 Flt1). This isoform upregulated the active form of Src and increased breast cancer cell invasiveness. Since expression of the transmembrane and soluble Flt1 isoforms of HUVEC is activated by Notch signaling, we wondered whether the expression of the intracellular isoform i21 Flt1 was also dependent on Notch activation. We report here that the expression of i21 Flt1 in HUVEC and MDA-MB-231 cells is downregulated by the γ-secretase inhibitor DAPT. In addition, treatment of MDA-MB-231 cells with siRNA specific for Notch-1 and Notch-3 downregulates the expression of i21 Flt1. In agreement with these findings, HUVEC and MDA-MB-231 breast cancer cells, cultured on dishes coated with recombinant human Dll4 extracellular domain, express higher levels of i21 Flt1. In cancer cells, Flt1 is a target of the micro RNA family miR-200. In MDA-MB-231 breast cancer cells, the truncated intracellular isoform i21 Flt1 is also negatively regulated by miR-200c. Retinoic acid interferes i21 Flt1 expression by downregulating Notch-3 and upregulating miR-200 expression. Treatment of MDA-MB-231 breast cancer cells with both a γ-secretase inhibitor and retinoic acid suppresses the expression of i21 Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach.

miR-200c: a versatile watchdog in cancer progression, EMT, and drug resistance

“…3). Notch also activates truncated isoform of Flt1/VEGFR-1 (which is downregulated in the presence of miR-200c [104] and of importance in angiogenesis-associated VEGF signaling) leading to activation of proto-oncogene Src, which cross talks with and downregulates miR-200c in an NF-κB/IL-6-dependent manner [105]. miR-200c regulates TLR4/MyD88-dependent activation of NF-κB by directly targeting MyD88.…”

Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

“…The i 21 VEGFR-1 protein is expressed in human endothelial cells and MDA-MB-breast cancer cells [23,24]. The human isoforms i 19 VEGFR-1 and i 28 VEGFR-1 are expressed in human testis (GenBank JF509744 and JF509745).…”

“…Protein i 21 VEGFR-1 was detected by Western blot analysis [23,24]. To confirm the specificity of the bands detected by the anti-VEGFR-1 antibody, we inhibited the expression of VEGFR-1 and i 21 VEGFR-1 by RNA interference.…”

“…Bands of 170 kD and 39 Kd, corresponding to the full-length transmembrane VEGFR-1 and the truncated intracellular isoform, respectively, disappear after RNA interference in human endothelial cells (HUVECs). Furthermore, the band of 39 kD, corresponding to i 21 Flt1, is no longer detectable after RNA interference of i 21 VEGFR-1 in MDA-MB-231 breast cancer cells [24]. …”

“…Expression of i 21 -VEGFR1, transcript and protein in MDA-MB-231 highly invasive breast cancer cells is controlled by the Notch signaling pathway [23,24]. Interference of the Notch signaling pathway by the inhibitor of γ-secretase DAPT decreases the expression of i 21 VEGFR-1 in MDA-MB-231cells [24].…”