Montserrat Pau

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

One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

SEVERAL NOVEL TRANSCRIPTS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE EXPRESSED IN ADULT CHICKEN TESTIS

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis. Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported. While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5′ region of the pre‐mRNA, resulting in at least six different types of mRNAs. The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock. These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis. J. Cell. Biochem. 71:127–139, 1998.

A novel intracellular isoform of VEGFR-1 activates Src and promotes cell invasion in MDA-MB-231 breast cancer cells.

Two types of VEGFR‐1 receptors have been characterized: a full‐length transmembrane receptor and a truncated extracellular soluble isoform (sVEGFR‐1). We report here the characterization, in normal and cancer cells, of a new family of intracellular isoforms of VEGFR‐1 resulting from alternative initiation of transcription in intronic sequences of the gene. While the classical isoforms of VEGFR‐1 were barely detectable in MDA‐MB‐231 breast cancer cells, one of the intracellular isoforms transcribed from intron 21 (i21VEGFR‐1) was the main isoform expressed in these cells. The new transcript encodes for a protein that contains only the phosphotransferase domain and the carboxyterminal tail of VEGFR‐1. Treatment of MDA‐MB‐231 cells with siRNA specific for the tyrosine domain of VEGFR‐1 suppressed the expression of i21VEGFR‐1, downregulated phosphorylation of Src at tyrosine 418, and reduced markedly the invasion capacity of these cells in vitro. Accordingly, overexpression of transfected i21VEGFR‐1 in MDA‐MB‐231 cells upregulated the active form of Src and increased invasiveness of MDA‐MB‐231 cells. The expression of i21VEGFR‐1 in MDA‐MB‐231 cells was inhibited by retinoic acid. Both, activation of Src and downregulation by retinoic acid, have been reported in other intracellular members of the Fms/Kit/PDGFR family of tyrosine kinases, particularly in the intracellular isoform of c‐kit, analogous structurally to i21VEGFR‐1 and frequently expressed in cancer cells. J. Cell. Biochem. 110: 732–742, 2010.

Down‐regulation of Flt‐1 gene expression by the proteasome inhibitor MG262

The mechanisms involved in the anti‐angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt‐1 (vascular endothelial growth factor receptor 1) was down‐regulated by the reversible proteasome inhibitor MG262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt‐1 by lipopolysaccharide (LPS) in macrophages and down‐regulated the expression of Flt‐1 after LPS induction. Flt‐1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt‐1, coding for a soluble form of the receptor (sFlt‐1) with anti‐angiogenic properties, was not down‐regulated in the same extent. Only a small decrease in the expression of VEGF and Ang‐2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang‐1. Since recent experiments have demonstrated the importance of anti‐Flt‐1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [ 2002 ]: Nat Med 8:831–840), our observation on down‐regulation of Flt‐1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation.

Four isoforms of the signal‐transduction and RNA‐binding protein QKI expressed during chicken spermatogenesis

Genes expressed during spermatogenesis undergo alternative initiation and alternative splicing and may be under the control of a coordinated mechanism of RNA processing. A family of proteins that combine features of signal‐transduction and RNA‐binding molecules could be instrumental in this process. We have characterized a cDNA from adult chicken testis that codifies a highly conserved member of the STAR protein family, the orthologue of the mouse quakinggene qkI. The predicted chicken protein differs only in four amino acids from the corresponding mouse protein. Messages of 7, 6, and 5 kb are expressed differentially during chicken spermatogenesis. The 5‐kb message, the predominant form in adult testis, presents heterogeneity in the coding region, showing insertions of 51 and 75 bp and a deletion of 24 bp, which gives rise to four possible isoforms of the protein. Mol. Reprod. Dev. 50:70–78, 1998.

Heat-shock inducible polyubiquitin gene UbI undergoes alternative initiation and alternative splicing in mature chicken testes.

Ubiquitin, a heat‐shock protein highly expressed during spermatogenesis, plays an essential role in the differentiation of the germinal cells, particularly in the structural changes of chromatin taking place at the end of the process. To shed light on the mechanisms that modulate transcriptional activity of the heat‐shock inducible polyubiquitin gene Ubl during spermatogenesis and stabilize the message when transcription is not longer active, we have compared the characteristics of Ubl transcripts in mature and immature testes and somatic cells. In mature chicken testes, transcription starts at a site placed closer to the heat‐shock promoters than in somatic tissues. This site is upstream from the TATA box used in somatic cells. In addition, Ubl transcript undergoes an alternative splicing that produces a longer 5′ untranslated region in mature testis. These findings may provide a basis for the observed increase in expression of Ubl in mature chicken testes and for the stability of the message when transcription ceases at the end of spermatogenesis. Mol. Reprod. Dev. 46:471–475, 1997.

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

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 (i21Flt1). 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 i21Flt1 was also dependent on Notch activation. We report here that the expression of i21Flt1 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 i21Flt1. 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 i21Flt1. 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 i21Flt1 is also negatively regulated by miR‐200c. Retinoic acid interferes i21Flt1 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 i21Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach. J. Cell. Biochem. 115: 52–61, 2014.

LoVo colon cancer cells resistant to oxaliplatin overexpress c-MET and VEGFR-1 and respond to VEGF with dephosphorylation of c-MET.

Oxaliplatin‐resistant LoVo colon cancer cells overexpressing c‐MET and VEGFR‐1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c‐MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin‐resistant cells (LoVoR) overexpress and phosphorylate c‐MET, upregulate the expression of transmembrane and soluble VEGFR‐1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, β‐catenin‐TCF4 and E‐cadherin. While c‐MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c‐MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c‐MET by VEGF is mediated by VEGFR‐1, since phosphorylation of c‐MET in the presence of VEGF is restored after silencing VEGFR‐1. Dephosphorylation of c‐MET by VEGF suggests that tumors coexpressing VEGFR‐1 and c‐MET may activate c‐MET as a result of anti‐VEGF therapy.

All-trans-retinoic acid activates the pro-invasive Src-YAP-Interleukin 6 axis in triple-negative MDA-MB-231 breast cancer cells while cerivastatin reverses this action

All-trans-retinoic acid (RA), the active metabolite of vitamin A, can reduce the malignant phenotype in some types of cancer and paradoxically also can promote cancer growth and invasion in others. For instance, it has been reported that RA induces tumor suppression in tumor xenografts of MDA-MB-468 breast cancer cells while increasing tumor growth and metastases in xenografts of MDA-MB-231 breast cancer cells. The signaling pathways involved in the pro-invasive action of retinoic acid remain mostly unknown. We show here that RA activates the pro-invasive axis Src-YAP-Interleukin 6 (Src-YAP-IL6) in triple negative MDA-MB-231 breast cancer cells, yielding to increased invasion of these cells. On the contrary, RA inhibits the Src-YAP-IL6 axis of triple-negative MDA-MB-468 cells, which results in decreased invasion phenotype. In both types of cells, inhibition of the Src-YAP-IL6 axis by the Src inhibitor PP2 drastically reduces migration and invasion. Src inhibition also downregulates the expression of a pro-invasive isoform of VEGFR1 in MDA-MB-231 breast cancer cells. Furthermore, interference of YAP nuclear translocation using the statin cerivastatin reverses the upregulation of Interleukin 6 (IL-6) and the pro-invasive effect of RA on MDA-MB-231 breast cancer cells and also decreases invasion and viability of MDA-MB-468 breast cancer cells. These results altogether suggest that RA induces pro-invasive or anti-invasive actions in two triple-negative breast cancer cell lines due to its ability to activate or inhibit the Src-YAP-IL6 axis in different cancer cells. The pro-invasive effect of RA can be reversed by the statin cerivastatin.