The role of a new CD44st in increasing the invasion capability of the human breast cancer cell line MCF-7

<p>Abstract</p> <p>Background</p> <p>CD44, a hyaluronan (HA) receptor, is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. The CD44 gene contains 20 exons that are alternatively spliced, giving rise to many CD44 isoforms, perhaps including tumor-specific sequences.</p> <p>Methods</p> <p>Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect CD44st mRNA and CD44 protein in sensitive MCF-7, Lovo, K562 and HL-60 cell lines as well as their parental counterparts, respectively. The full length cDNA encoding CD44st was obtained from the total RNA isolated from MCF-7/Adr cells by RT-PCR, and subcloned into the pMD19-T vector. The CD44st gene sequence and open reading frame were confirmed by restriction enzyme analysis and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44st was transfected into MCF-7 cells using Lipofectamine. After transfection, the positive clones were obtained by G418 screening. The changes of the MMP-2 and MMP-9 genes and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells penetrating through the artificial matrix membrane in each group (MCF-7, MCF-7+HA, MCF-7/neo, MCF-7/neo+HA, MCF-7/CD44st, MCF-7/CD44st+HA and MCF-7/CD44st+Anti-CD44+HA) was counted to compare the change of the invasion capability regulated by the CD44st. Erk and P-Erk were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by the CD44st.</p> <p>Results</p> <p>Sensitive MCF-7, Lovo, K562 and HL-60 cells did not contain CD44st mRNA and CD44 protein. In contrast, the multidrug resistance MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells expressed CD44st mRNA and CD44 protein. The CD44st mRNA gene sequence was successfully cloned into the recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the gene sequence of CD44st that was composed of exons 1 to 4, 16 to 17, and 1 to 205 bases of exons 18. The new gene sequence was sent to NCBI for publication, and obtained the registration number FJ216964. The up-regulated level of the mRNA of the CD44 gene and the CD44 protein were detected, respectively, by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44st. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by HA treatment, and blocked by CD44 neutralizing antibody. MCF-7/CD44st cells pretreated with the neutralizing antibody against CD44, and the inhibitor of MAPKs signaling pathway, could strongly block the expression of P-Erk.</p> <p>Conclusions</p> <p>A new CD44st was expressed in multidrug resistant MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells. The expression vector pcDNA3.1-CD44st was cloned and constructed successfully, and stably transfected into MCF-7 cells. HA could interact with the new CD44st and regulate the expression of MMP-2 and MMP-9, which could increase the invasion capability of MCF-7 cells through the Ras/MAPK signaling pathway.</p

Relationship of circulating hyaluronic Acid levels to disease control in asthma and asthmatic pregnancy.

Uncontrolled asthma is a risk factor for pregnancy-related complications. Hyaluronic acid (HA), a potential peripheral blood marker of tissue fibrosis in various diseases, promotes eosinophil survival and plays a role in asthmatic airway inflammation as well as in physiological processes necessary to maintain normal pregnancy; however the level of circulating HA in asthma and asthmatic pregnancy is unknown. We investigated HA levels in asthmatic patients (N = 52; asthmatic pregnant (AP) N = 16; asthmatic non-pregnant (ANP) N = 36) and tested their relationship to asthma control. Serum HA level was lower in AP than in ANP patients (27 [24.7-31.55] vs. 37.4 [30.1-66.55] ng/mL, p = 0.006); the difference attenuated to a trend after its adjustment for patients' age (p = 0.056). HA levels and airway resistance were positively (r = 0.467, p = 0.004), HA levels and Asthma Control Test (ACT) total score inversely (r = -0.437, p = 0.01) associated in ANP patients; these relationships remained significant even after their adjustments for age. The potential value of HA in the determination of asthma control was analyzed using ROC analysis which revealed that HA values discriminate patients with ACT total score >/=20 (controlled patients) and <20 (uncontrolled patients) with a 0.826 efficacy (AUC, 95% CI: 0.69-0.97, p = 0.001) when 37.4 ng/mL is used as cut-off value in ANP group, and with 0.78 efficacy (AUC, 95% CI: 0.65-0.92, p = 0.0009) in the whole asthmatic cohort. In conclusion circulating HA might be a marker of asthma control, as it correlates with airway resistance and has good sensitivity in the detection of impaired asthma control. Decrease of HA level in pregnancy may be the consequence of pregnancy induced immune tolerance

Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.Carmela Ricciardelli, Miranda P Ween, Noor A Lokman, Izza A Tan, Carmen E Pyragius, and Martin K Oehle

A pivotal role for galectin-1 in fetomaternal tolerance.

International audienceA successful pregnancy requires synchronized adaptation of maternal immune-endocrine mechanisms to the fetus. Here we show that galectin-1 (Gal-1), an immunoregulatory glycan-binding protein, has a pivotal role in conferring fetomaternal tolerance. Consistently with a marked decrease in Gal-1 expression during failing pregnancies, Gal-1-deficient (Lgals1(-/-)) mice showed higher rates of fetal loss compared to wild-type mice in allogeneic matings, whereas fetal survival was unaffected in syngeneic matings. Treatment with recombinant Gal-1 prevented fetal loss and restored tolerance through multiple mechanisms, including the induction of tolerogenic dendritic cells, which in turn promoted the expansion of interleukin-10 (IL-10)-secreting regulatory T cells in vivo. Accordingly, Gal-1's protective effects were abrogated in mice depleted of regulatory T cells or deficient in IL-10. In addition, we provide evidence for synergy between Gal-1 and progesterone in the maintenance of pregnancy. Thus, Gal-1 is a pivotal regulator of fetomaternal tolerance that has potential therapeutic implications in threatened pregnancies

MiR-16 mediates trastuzumab and lapatinib response in ErbB-2-positive breast and gastric cancer via its novel targets CCNJ and FUBP1

ErbB-2 amplification/overexpression accounts for an aggressive breast cancer (BC) subtype (ErbB-2-positive). Enhanced ErbB-2expression was also found in gastric cancer (GC) and has been correlated with poor clinical outcome. The ErbB-2-targeted therapiestrastuzumab (TZ), a monoclonal antibody, and lapatinib, a tyrosine kinase inhibitor, have proved highly beneficial. However,resistance to such therapies remains a major clinical challenge. We here revealed a novel mechanism underlying theantiproliferative effects of both agents in ErbB-2-positive BC and GC. TZ and lapatinib ability to block extracellular signal-regulatedkinases 1/2 and phosphatidylinositol-3 kinase (PI3K)/AKT in sensitive cells inhibits c-Myc activation, which results in upregulation ofmiR-16. Forced expression of miR-16 inhibited in vitro proliferation in BC and GC cells, both sensitive and resistant to TZ andlapatinib, as well as in a preclinical BC model resistant to these agents. This reveals miR-16 role as tumor suppressor in ErbB-2-positive BC and GC. Using genome-wide expression studies and miRNA target prediction algorithms, we identified cyclin J and farupstream element-binding protein 1 (FUBP1) as novel miR-16 targets, which mediate miR-16 antiproliferative effects. Supportingthe clinical relevance of our results, we found that high levels of miR-16 and low or null FUBP1 expression correlate with TZresponse in ErbB-2-positive primary BCs. These findings highlight a potential role of miR-16 and FUBP1 as biomarkers of sensitivityto TZ therapy. Furthermore, we revealed miR-16 as an innovative therapeutic agent for TZ- and lapatinib-resistant ErbB-2-positiveBC and GC.Fil: Venturutti, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cordo Russo, Rosalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rivas, Martin Alfredo. Cornell University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Izzo, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Oakley, R. H.. National Institutes of Health; Estados UnidosFil: Pereyra, M. G.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: De Martino, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Proietti Anastasi, Cecilia Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Investigación En Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Roa, J. C.. Universidad de la Frontera. Nucleo Cientifico y Tecnológico en Recursos Naturales; Chile. Pontificia Universidad Católica de Chile; ChileFil: Guzmán, P.. Universidad de la Frontera. Nucleo Cientifico y Tecnológico en Recursos Naturales; ChileFil: Cortese, E.. Instituto Universitario Aeronautico. Instituto Nacional de Derecho Aeronautico y Espacial; ArgentinaFil: Allemandi, Daniel Alberto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Huang, T. H.. University of Texas; Estados UnidosFil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cidlowski, J. A.. National Institutes of Health; Estados UnidosFil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin