Myriam Sánchez-Gómez

Differential expression of IGF-I and insulin receptor isoforms in HPV positive and negative human cervical cancer cell lines.

Human papillomavirus (HPV) is the main risk factor for cervical cancer; however, some carcinomas occur in the absence of the virus. IGF-IR and an isoform of the insulin receptor, IR-A, play important roles in cancer. In this study we assessed the role of the IGF/insulin receptors in cervical cancer cell lines with different HPV status, SiHa (HPV positive), and C33a (HPV negative). Different patterns of receptor expression were found; while SiHa expressed IGF-IR, IR-A and IR-B, and IR/IGF-IR hybrid receptors, C33a cells expressed the IR-A only. Tyrosine phosphorylation of these receptors in response to their corresponding ligands correlated with the expression level of these receptors in the cell lines. Activation of PI3-K and MAPK pathways was revealed in both cell lines, however, no effects on proliferation, migration, or invasion were observed. Here we show that cervical cancer cell lines--positive and negative for HPV--differ in the type of insulin and IGF-1 receptors expressed. Additional studies are needed for characterization of the role of IR-A in cervical carcinogenesis.

T-Cell Populations and Cytokine Expression Are Impaired in Thymus and Spleen of Protein Malnourished BALB/c Mice Infected with Leishmania infantum

Visceral leishmaniasis (VL) is a parasitic infectious disease that causes significant morbidity and mortality in the tropical and subtropical regions of the world. Although infections with visceralizing Leishmania may be asymptomatic, factors such as undernutrition increase the likelihood of progressing to clinical disease. Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered as a primary risk factor for the development of clinical VL. However, data regarding the immunological basis of this association are scarce. With the aim to analyze the effects of protein malnutrition on Leishmania infantum infection, we used BALB/c mice subjected to control or low protein isocaloric diets. Each animal group was divided into two subgroups and one was infected with L. infantum resulting in four study groups: animals fed 14% protein diet (CP), animals fed 4% protein diet (LP), animals fed 14% protein diet and infected (CPi), and animals fed 4% protein diet and infected (LPi).The susceptibility to L. infantum infection and immune responses were assessed in terms of body and lymphoid organ weight, parasite load, lymphocyte subpopulations, and cytokine expression. LPi mice had a significant reduction of body and lymphoid organ weight and exhibited a severe decrease of lymphoid follicles in the spleen. Moreover, LPi animals showed a significant decrease in CD4+CD8+ T cells in the thymus, whereas there was an increase of CD4+ and CD8+ T cells percentages in the spleen. Notably, the cytokine mRNA levels in the thymus and spleen of protein malnourished-infected animals were altered compared to the CP mice. Protein malnutrition results in a drastic dysregulation of T cells and cytokine expression in the thymus and spleen of L. infantum-infected BALB/c mice, which may lead to defective regulation of the thymocyte population and an impaired splenic immune response, accelerating the events of a normal course of infection.

Simvastatin impairs growth hormone-activated signal transducer and activator of transcription (STAT) signaling pathway in UMR-106 osteosarcoma cells

Recent studies have demonstrated that statins reduce cell viability and induce apoptosis in various types of cancer cells. The molecular mechanisms underlying these effects are poorly understood. The JAK/STAT pathway plays an important role in the regulation of proliferation and apoptosis in many tissues, and its deregulation is believed to be involved in tumorigenesis and cancer. The physiological activation of STAT proteins by GH is rapid but transient in nature and its inactivation is regulated mainly by the expression of SOCS proteins. UMR-106 osteosarcoma cells express a GH-responsive JAK2/STAT5 signaling pathway, providing an experimental model to study the influence of statins on this system. In this study we investigated the actions of simvastatin on cell proliferation, migration, and invasion on UMR-106 cells and examined whether alterations in GH-stimulated JAK/STAT/SOCS signaling may be observed. Results showed that treatment of osteosarcoma cells with simvastatin at 3 to 10 µM doses decreases cell proliferation, migration, and invasion in a time- and dose-dependent manner. At the molecular level, although the mechanisms used by simvastatin are not entirely clear, the effect of the statin on the reduction of JAK2 and STAT5 phosphorylation levels may partially explain the decrease in the GH-stimulated STAT5 transcriptional activity. This effect correlated with a time- and dose-dependent increase of SOCS-3 expression levels in cells treated with simvastatin, a regulatory role that has not been previously described. Furthermore, the finding that simvastatin is capable of inducing SOCS-3 and CIS genes expression shows the potential of the JAK/STAT pathway as a therapeutic target, reinforcing the efficacy of simvastatin as chemotherapeutic drug for the treatment of osteosarcoma.

Regulation of the splenic somatotropic axis by dietary protein and insulin-like growth factor-I in the rat

Protein intake is a critical regulatory factor of the GH/IGF-I axis. Recently, it has been shown that splenic GH/IGF-I may respond to nutritional stress by preserving tissue homeostasis. To study the effects of exogenous administration of rhIGF-I on the splenic GH/IGF-I axis in protein malnourished rats, six-week-old male rats were assigned to one of four isocaloric diets differing in the protein content (0%, 4%, 12% and 20%) for a period of 12 days. Animals in the same dietary group on day 5 were randomly divided into two groups and during 7 days received a continuous subcutaneous infusion of either vehicle or rhIGF-I (300 microg/day). A low protein intake decreased the circulating levels of IGF-I, IGFBP-3, GH and insulin whereas the serum levels of IGFBP-1 were increased. Splenic IGFBP-3, -4 and -6 mRNA expression were up-regulated by protein malnutrition. Similarly, IGF-IR and GHR mRNA expression were significantly increased by the lack of dietary protein, whereas the levels of IGF-I mRNA remained unchanged. Exogenous rhIGF-I administration increased the circulating levels of IGFBP-1 and -3 in protein malnourished rats and reduced significantly the GH and insulin levels in well-fed rats. Similarly, rhIGF-I increased significantly the expression of the GHR in the spleen and splenic weight in all dietary groups, whereas nitrogen balance was enhanced only in the high-protein diet group. Among the cell subpopulations, B lymphocytes showed the highest GHR expression. These results suggest that in catabolic stress, induced by protein malnutrition the splenic GH/IGF-I axis is an important modulator and contributes to the maintenance of the homeostasis of the immune system.

Cervical scrapes levels of insulin-like growth factor-II and insulin-like growth factor binding protein 3 in women with squamous intraepithelial lesions and cervical cancer.

A growing number of studies have demonstrated an association between serum levels of insulin-like growth factors (IGFs) and IGF binding protein-3 (IGFBP-3) and increased risk for various cancers. The aim of this study was to evaluate the relationship between levels of IGF-II or IGFBP-3 in cervical scrapes with cervical cancer and precancerous lesions: low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL). 4 groups of cases were examined: LSIL (n=20), HSIL (n=28), cervical cancer (n=45), and controls (n=51). Control subjects were women with normal, HPV DNA-negative Papanicolau (Pap) test. IGF-II and IGFBP-3 levels in cervical scrapes were measured by ELISA. Results show that median protein levels of IGF-II were significantly lower in cervical cancer cases vs. controls (446.5 ng/mg vs. 1,168.6 ng/mg, p<0.001). Significantly higher values of IGFBP-3 were found in HSIL vs. controls (median: 549.5 ng/mg vs. 216 ng/mg; p=0.018), and were not affected by HR HPV infection, meanwhile no significant differences were observed in IGFBP-3 levels between LSIL or cervical cancer as compared to controls. These data suggests that the progression to cervical cancer is associated with alterations in the IGF system and not affected by HR HPV infection. More studies are needed to understand the possible role of IGFBP-3 in cervical carcinogenesis.

Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals.

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.

New Biomarkers for Cervical Cancer – Perspectives from the IGF System

The insulin-like growth factor (IGF) family is organized in a complex regulatory network at the cellular and sub-cellular levels. In the human the IGF system has a key physiological role in the development of the organism and maintenance of normal cellular function during foetal and postnatal life. The IGF system consists of three ligands, IGF-I, IGF-II and Insulin; three cell membrane receptors, IGF-I receptor (IGF-IR), insulin receptor (IR), and IGF-II receptor (IGF-IIR); six high-affinity IGF binding proteins, IGFBP-1 through -6, their specific proteases (IGFBP proteases) and membrane receptors (IGFBP-R) (Fig. 1).

Abstract B23: Colombians with high percentage of African ancestry, carrying a specific IL1B haplotype, have increased risk of colorectal cancer

Background: Colorectal cancer mortality rates are heterogeneous across Colombian territory and this variation could be explained by regional differences in Colombian ethnicity due to our high population admixture. Different frequencies of the interleukin 1 beta ( IL1B ) genetic variations have been described among diverse ethnical groups, but there are no reports on the possible association of these variants with colorectal cancer (CRC) risk as an effect of the genetic population dynamics in Colombia. Methods: To evaluate the association of IL1B haplotypes with colorectal carcinogenesis, we genotyped 317 CRC cases, 199 adenomatous polyps (AP) and 511 age-sex matched controls recruited from six Colombian cities located either on the coastal or the inner zone of the country. Five IL1B SNPs (-3737, -1464, -511, -31 and +3954) were genotyped and all were in HWE (p>0.05). We used data from previous analysis to estimate individual ancestral genetic fractions using STRUCTURE software based on allele frequencies of 678 autosomal SNPs cross-matched with the HapMap dataset, not in LD, and assuming three distinct population origins (CEU, LWJ and CHB populations) from HapMap project as references. Results: We found a 4-SNPs haplotype block (-3737C/-1464G/-511T/-31C) with high LD associated with CRC risk. Additional analysis revealed that this association persisted after adjusting by confounding variables including ancestry information (OR 1.95; CI 1.12 - 3.38; p =0.02). Interestingly, this association was evident among individuals from the coastal zone of the country and it was related with their higher African ancestry fraction (OR 2.26; 95%CI 1.22-4.18; p =0.01). Our results are in agreement with previous reports showing that this haplotype increase the IL1B promoter activity and that is highly prevalent in African-Americans. Conclusions: To the best of our knowledge, this is the first study seeking for the association between CRC risk and IL1B gene in a haplotype context in a population-based association study in Colombia, an admixed population. Our results represent consistent evidence on how the identification of genetic biomarkers of susceptibility could be used to discriminate populations at increased risk of CRC development. Citation Format: Maria Carolina Sanabria-Salas, Gustavo Hernandez-Suarez, Adriana Umana-Perez, Martha Lucia Serrano-Lopez, Myriam Sanchez-Gomez, Martha Patricia Rojas, Jovanny Zabaleta. Colombians with high percentage of African ancestry, carrying a specific IL1B haplotype, have increased risk of colorectal cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B23. doi:10.1158/1538-7755.DISP13-B23

OR4,24 A phosphoproteomic view of the IGF-II signaling

gene or in ‘small for gestational age’ infants are associated with increased risk of developing a metabolic syndrome. Additionally, recent studies also imply the use of the IGF-1R as a target tool to study insulin resistance and Type 2 diabetes. Our lab has used the Igf1r mice to unravel the mechanism of disrupted glucose metabolism caused by IGF-1R haploinsufficiency. The results demonstrate a consistent reduction in the IGF-1 signaling in the muscle tissue of Igf1r mice. The aged male Igf1r mice, in particular, exhibit glucose and insulin intolerance, fasting hyperinsulinemia, reduced pGSK3 levels and an increased expression of lipogenic enzymes, a signature of metabolic disorder. This study proposes a new mechanism whereby reduced IGF-1 signaling results in hyperglycemia and hyperinsulinemia, a secondary consequence of which is ectopic fat accretion in the muscle and liver. An intense metabolic profiling of this model will be imminent in the understanding of molecular mechanisms favoring insulin resistance, thereby leading to the development of novel therapies for Type 2 diabetes.