Pau Mezquita

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.

B Lymphocyte-Specific c-Myc Expression Stimulates Early and Functional Expansion of the Vasculature and Lymphatics during Lymphomagenesis

Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitts lymphoma (E micro -c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) E micro -c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in E micro -c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from E micro -c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively.

Carnitine palmitoyltransferase 1C deficiency causes motor impairment and hypoactivity

Carnitine palmitoyltransferase 1c (CPT1C), a brain-specific protein localized in the endoplasmic reticulum of neurons, is expressed in almost all brain regions, but its only known functions to date are involved in the hypothalamic control of energy homeostasis and in hippocampus-dependent spatial learning. To identify other physiological and behavioral functions of this protein, we performed a battery of neurological tests on Cpt1c-deficient mice. The animals showed intact autonomic and sensory systems, but some motor disturbances were observed. A more detailed study of motor function revealed impaired coordination and gait, severe muscle weakness, and reduced daily locomotor activity. Analysis of motor function in these mice at ages of 6-24 weeks showed that motor disorders were already present in young animals and that impairment increased progressively with age. Analysis of CPT1C expression in different motor brain areas during development revealed that CPT1C levels were low from birth to postnatal day 10 and then rapidly increased peaking at postnatal day 21, which suggests that CPT1C plays a relevant role in motor function during and after weaning. As CPT1C is known to regulate ceramide levels, we measured these biolipids in different motor areas in adult mice. Cerebellar, striatum, and motor cortex extracts from Cpt1c knockout mice showed reduced levels of ceramide and its derivative sphingosine when compared to wild-type animals. Our results indicate that altered ceramide metabolism in motor brain areas induced by Cpt1c deficiency causes progressive motor dysfunction from a young age.

NOD/SCID repopulating cells contribute only to short-term repopulation in the baboon.

We have previously compared the repopulation ability of gene-modified baboon CD34+ cells in an autologous transplantation versus a xenotransplant model in irradiated nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Baboon CD34-selected marrow cells were transduced with a gammaretrovirus vector and infused into irradiated baboons and NOD/SCID mice. A limited integration-site analysis could only detect two common retrovirus integration sites in the NOD/SCID and monkey. Here, we performed locus-specific PCR on 30 clones recovered from NOD/SCID β2-microglobulin mice reconstituted with transduced baboon CD34+ cells. We identified five common integrants in the baboon early after transplant (2–6 weeks) but none during the long-term follow-up (6 and 12 months). These results confirm that repopulating cells in the NOD/SCID mouse contribute only to short-term repopulation in a clinically relevant large animal model.

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.

The atypical cyclin CNTD2 promotes colon cancer cell proliferation and migration

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8–10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF. Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.

5. Differential Expression of PIT1 and PIT2 after G-CSF Mobilization Is Associated with Efficient Gene Transfer Using GALV-Pseudotyped Gammaretroviral Vectors

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF), however, SCF is unavailable for clinical use. A recent study of a competitive repopulation assay in the rhesus macaque model showed very inefficient engraftment of transduced G-CSF-mobilized peripheral blood (G-PBSC) CD34+ cells relative to G-CSF- and SCF-mobilized cells using amphotropic pseudotypes (Hematti P et al Blood 101, 2003, 2199|[ndash]|2205). Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient engraftment of transduced G-PBSC in our baboon model using Phoenix-GALV pseudotype vectors. In order to better compare these results to those from previous experiments utilizing G-CSF- and SCF-primed bone marrow (G&S-BM) as a stem cell source, the first two baboons also received G-CSF-primed BM (G-BM) in a competitive repopulation. We transplanted three baboons with G-CSF-mobilized CD34+ cells transduced with GALV pseudotyped retroviral vectors. We observed high-level, persistent engraftment of transduced G-PBSC in all three animals with gene marking in granulocytes in one animal up to 60%. To determine retrovirus integration pattern of clones that contributed to long-term hematopoiesis linear amplification mediated (LAM)-PCR was used for all animal samples studied. In all animals analyzed multiple clones could be detected and in animals that received both G-PBSC and G-BM multiple clones contributed from both transduced cell populations. These in vivo marking results were not in agreement with the report by Hematti et al so we sought to determine whether the different results might be explained by the fact that different combinations of cytokines (G-CSF and SCF) for mobilization modulate receptor expression of the amphotropic (PIT2) and GALV (PIT1). Analysis of receptor expression in baboon CD34+ cells after administration of different combinations of cytokines for mobilization found receptor expression was dramatically affected by different mobilization regimens with G-CSF and SCF increasing PIT2 expression and G-CSF alone increasing PIT1 expression. The low in vivo gene marking in previous reports using amphotropic pseudotyped vectors and G-CSF alone for mobilization could be explained because in animals treated with G-CSF alone, at least in baboons, the amphotropic receptor expression was minimal while including SCF in the mobilization regimen substantially increased amphotropic receptor expression. In contrast to other studies, our high in vivo marking in G-CSF-mobilized baboon peripheral blood CD34+ cells demonstrates the feasibility of efficiently transducing G-PBSC in clinical gene therapy trials and verifies that there is no inherent stem cell deficiency in G-PBSC mobilized cells.