Predictive Outcomes for HER2-enriched Cancer Using Growth and Metastasis Signatures Driven By SPARC.

Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer.Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR.

CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells.

Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-α, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. In the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site.

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus).

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERß. Yet throughout pregnancy, ERα and ERß levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.

Integrin-linked kinase (ILK) expression correlates with tumor severity in clear cell renal carcinoma.

Integrin-linked kinase (ILK) is an unique intracellular serine/threonine kinase and adapter protein. When dysregulated, it has been associated with increased cell proliferation, anchorage-independent cell growth, evasion of apoptosis, angiogenesis, invasion of surrounding tissues, downregulation of E-cadherin expression, nuclear translocation of ß-catenin and metastasis, all features of tumoral malignancy. The objective of the present work was to evaluate the expression of ILK in clear cell renal carcinomas (CCRC) as a possible prognostic indicator. ILK immunoexpression was evaluated in a tissue microarray (TMA) with 45 human CCRCs. In addition, the apoptotic and proliferative indices and the immuno-expression of ß-catenin and E-cadherin were also evaluated. E-cadherin expression was significantly decreased in tumors with positive ILK expression in relation to those with negative immunoexpression (p = 0.011). ILK immunostaining was significantly increased in high-grade in comparison to low-grade CCRCs (p = 0.0008). ILK expression was also associated with increased proliferative index (p = 0.020), tumor size >7.0 cm (p = 0.018) and with renal vein and capsule invasion (p = 0.003 and p = 0.00). Finally, tumors stage I and II (noninvasive) presented significantly reduced ILK immunoexpression when compared to stage III (locally invasive) (p = 0.0028). ILK immunoexpression in CCRC increases with loss of intercellular adhesion, nuclear grading, increased proliferative index and Robson stage. Altogether, our data suggest a possible role for ILK in the progression of CRCC.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

Proliferation of seminiferous epithelium cells during the postnatal development in goats.

The objective of this research study was to evaluate the proliferation of somatic and germ cells in the seminiferous epithelium of Alpine goats. A total of 47 goats reared in semi-intensive conditions were used, divided into age groups from birth up to 12 months of age. Following castration, testis fragments were included in plastic resin and were mounted in Entellan(®) for histometric evaluations. In general, the total number of germ cells per seminiferous cord transverse section was low until 4 months of age. An increase was observed between 4 and 5 months, lasting until 9 months of age. From that age on, this number tended to stabilize, until 12 months. The population of support cells (undifferentiated support cells and Sertoli cells) remained constant from birth to the first month, when it peaked. This was followed by a reduction until the fifth month of age. From that age on, there was differentiation in mature Sertoli cells, and its population remained constant until 12 months of age. It can be concluded that Alpine goats were in the impuberal phase from birth to 3 months of age, prepuberal phase during the fourth month, reached puberty at 5 months of age, postpuberal phase from 6 to 8 months, and reached sexual maturity at 9 months of age. Overall yield of spermatogenesis and Sertoli cell index increased from puberty up to the 12th month of age.

Tumor-specific MAGE proteins as regulators of p53 function.

Since its discovery in 1991, the knowledge about the tumor specific melanoma antigen gene (MAGE-I) family has been continuously increasing. Initially, MAGE-I proteins were considered as selective targets for immunotherapy. More recently, emerging data obtained from different cellular mechanisms controlled by MAGE-I proteins suggest a key role in the regulation of important pathways linked to cell proliferation. This is in part due to the ability of some MAGE-I proteins to control the p53 tumor suppressor. In this review, we focus on the mechanisms proposed to explain how MAGE-I proteins affect p53 functions.

Antagonic activities of Trypanosoma cruzi metacaspases affect the balance between cell proliferation, death and differentiation.

Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.

MEIS1, PREP1, and PBX4 are differentially expressed in acute lymphoblastic leukemia: association of MEIS1 expression with higher proliferation and chemotherapy resistance.

BACKGROUND: The Three-amino acid-loop-extension (TALE) superfamily of homeodomain-containing transcription factors have been implicated in normal hematopoiesis and in leukemogenesis and are important survival, differentiation, and apoptosis pathway modulators. In this work, we determined the expression levels of TALE genes in leukemic-derived cell lines, in blood samples of patients with Acute lymphoblastic leukemia (ALL), and in the blood samples of healthy donors. RESULTS: Here we show increased expression of MEIS1, MEIS2, and PREP1 genes in leukemia-derived cell lines compared with blood normal cells. High levels of MEIS1 and PREP1, and low levels of PBX4 expression were also founded in samples of patients with ALL. Importantly, silencing of MEIS1 decreases the proliferation of leukemia-derived cells but increases their survival after etoposide treatment. Etoposide-induced apoptosis induces down-regulation of MEIS1 expression or PREP1 up-regulation in chemotherapy-resistant cells. CONCLUSIONS: Our results indicate that up-regulation of MEIS1 is important for sustaining proliferation of leukemic cells and that down-regulation of MEIS1 or up-regulation of PREP1 and PBX genes could be implicated in the modulation of the cellular response to chemotherapeutic-induced apoptosis.

Role of H4 receptor in histamine-mediated responses in human melanoma.

We have previously reported that histamine at micromolar concentrations reduces the proliferation of melanoma cell lines. It is also known that melanoma cells express histamine H1, H2, and H3 receptors. The aim of this study was to investigate the presence of histamine H4 receptor (H4R) in human melanoma cells and its associated biological processes. To better understand the importance of histamine in tumor development, we explored the expression of H4R in human melanoma tissue biopsies. The expression of H4R in WM35 and M1/15 cells was analyzed by reverse-transcription-PCR, western blot, and immunocytochemistry. To characterize the biological responses we evaluated cell proliferation by clonogenic assay and 5-bromo-2'-deoxyuridine incorporation. In addition, cell senescence and differentiation were determined by ß-galactosidase enzyme assay and dopa oxidase activity, respectively. The expression levels of H4R were determined by immunohistochemistry in 19 samples of human malignant lesions. Results indicate that melanoma cells express H4R at the messenger RNA and protein levels. By using histamine agonists, antagonists, and H4R small-interfering RNA we showed that the inhibitory effect of histamine on proliferation was in part mediated through the stimulation of the H4R. The decrease in proliferation was associated with an induction of cell senescence and an increase in melanogenesis, which is a differentiation marker of these cells. Furthermore, H4R was expressed in 42% of human melanoma biopsies. To our knowledge, this is the first report that describes the presence of the H4R in melanoma cells and tissue, suggesting a potential therapeutic application of H4R ligands.

Adaptation of the "Dynamic Method" for measuring the specific respiration rate in oxygen transfer systems through diffusion membrane.

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.

Cell proliferation and cytoarchitectural remodeling during spinal cord reconnection in the fresh-water turtle Trachemys dorbignyi.

In fresh-water turtles, the bridge connecting the proximal and caudal stumps of transected spinal cords consists of regenerating axons running through a glial cellular matrix. To understand the process leading to the generation of the scaffold bridging the lesion, we analyzed the mitotic activity triggered by spinal injury in animals maintained alive for 20-30 days after spinal cord transection. Flow cytometry and bromodeoxyuridine (BrdU)-labeling experiments revealed a significant increment of cycling cells around the lesion epicenter. BrdU-tagged cells maintained a close association with regenerating axons. Most dividing cells expressed the brain lipid-binding protein (BLBP). Cells with BrdU-positive nuclei expressed glial fibrillary acidic protein. As spinal cord regeneration involves dynamic cell rearrangements, we explored the ultra-structure of the bridge and found cells with the aspect of immature oligodendrocytes forming an embryonic-like microenvironment. These cells supported and ensheathed regenerating axons that were recognized by immunocytological and electron-microscopical procedures. Since functional recovery depends on proper impulse transmission, we examined the anatomical axon-glia relationships near the lesion epicenter. Computer-assisted three-dimensional models revealed helical axon-glial junctions in which the intercellular space appeared to be reduced (5-7 nm). Serial-sectioning analysis revealed that fibril-containing processes provided myelinating axon sheaths. Thus, disruption of the ependymal layer elicits mitotic activity predominantly in radial glia expressing BLBP on the lateral aspects of the ependyma. These cycling cells seem to migrate and contribute to the bridge providing the main support and sheaths for regenerating axons.

Enhanced neural progenitor/stem cells self-renewal via the interaction of stress-inducible protein 1 with the prion protein.

Prion protein (PrP(C) ), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C) -STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+) ) and PrP(C) -null (Prnp(0/0) ) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C) , with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development.

Magnetic field-magnetic nanoparticle culture system used to grow in vitro murine embryonic stem cells.

The in vitro growth of embryonic stem cells (ESCs) is usually obtained in the presence of murine embryonic fibroblasts (MEF), but new methods for in vitro expansion of ESCs should be developed due to their potential clinical use. This study aims to establish a culture system to expand and maintain ESCs in the absence of MEF by using murine embryonic stem cells (mECS) as a model of embryonic stem cell. Magnetic nanoparticles (MNPs) were used for growing mESCs in the presence of an external magnetic field, creating the magnetic field-magnetic nanoparticle (MF-MNP) culture system. The growth characteristics were evaluated showing a doubling time slightly higher for mESCs cultivated in the presence of the system than in the presence of the MEF. The undifferentiated state was characterized by RT-PCR, immunofluorescence, alkaline phosphatase activity and electron microscopy. Murine embryonic stem cells cultivated in presence of the MF-MNP culture system exhibited Oct-4 and Nanog expression and high alkaline phosphatase activity. Ultrastructural morphology showed that the MF-MNP culture system did not interfere with processes that cause structural changes in the cytoplasm or nucleus. The MF-MNP culture system provides a tool for in vitro expansion of mESCs and could contribute to studies that aim the therapeutic use of embryonic stem cells.

Tyrosine kinase A receptor (trkA): a potential marker in epithelial ovarian cancer.

OBJECTIVES: To evaluate the role of trkA receptor as a potential tumor marker in serous epithelial ovarian cancer and its relationship with the angiogenic factors expression as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Additionally, to examine whether NGF and VEGF secreted by epithelial ovarian cancer (EOC) explants and from epithelial ovarian cancer cell line (A2780) are involved in the process of angiogenesis, such as cellular proliferation, migration and differentiation of the human endothelial cell line (EA.hy926). METHODS: The mRNA levels of VEGF, NGF and trkA receptors were measured using PCR in 60 ovarian samples. Cellular localization and semi-quantitative estimation of VEGF, NGF, total trkA and p-trkA was performed using IHC in epithelial cells. NGF, total trkA and p-trkA protein were also evaluated in endothelial cells from the same tissues. Human endothelial cell line EA.hy926 was cultured with conditioned media obtained from both EOC explants and from the A2780 cell line, with or without NGF stimulus. RESULTS: Significantly higher levels of NGF, total trkA and p-trkA protein expressions were observed in epithelial and endothelial cells in poorly differentiated EOC versus normal ovary. Interestingly, the p-trkA receptor expression level showed the most significant difference and its presence was only found in borderline tumor and EOC samples indicating the importance of trkA receptor in EOC as a potential tumor marker. A significant increase in proliferation, migration and differentiation of EA.hy926 cells was observed with NGF, and this effect was significantly reverted when NGF was immuno-blocked and when a trkA inhibitor was used, showing that NGF is an important angiogenic factor in EOC by activating its trkA receptor. CONCLUSION: These results indicate that p-trkA may be considered as a new potential tumor marker in EOC, and that NGF may also act as a direct angiogenic factor in EOC.

Acute inflammation promotes early cellular stimulation of the epithelial and stromal compartments of the rat prostate.

BACKGROUND: It has been proposed that prostatic inflammation plays a pivotal role in the pathophysiology of benign hyperplasia and prostate cancer. However, little information is available about the prostatic reaction to bacterial compounds in vivo. Our aim was therefore to evaluate the early effects of bacterial infection on rat ventral prostate compartments. METHODS: Using a rat model of acute bacterial prostatitis by Escherichia coli, we analyzed the histological and ultrastructural changes in the prostate at 24, 48, and 72 hr postinfection. Prostatic tissues were immunostained for prostatic binding protein (PBP), ACTA2, ErbB1, and ErbB2 receptors, TUNEL, and markers of cell proliferation. Dot and Western blots for PBP, ACTA2, ErbB1, ErbB2, and TGFbeta1 were also performed. RESULTS: The prostatic epithelium became hypertrophied, with increases in PBP and ErbB1 expression at 24 hr postinfection. Moreover, inflammation induced the expression of ErbB2, a receptor strongly involved in carcinogenesis. These alterations were more pronounced at 48 hr, but the epithelium also showed apoptosis and finally atrophy at 72 hr postinfection, with a decrease in PBP and ErbB receptors. Interestingly, the epithelial cells exhibited a high level of proliferation in response to the bacteria. The stromal reaction to acute inflammation was initially characterized by smooth muscle hypertrophy. Afterwards, muscle cells acquired a secretory phenotype, with a reduction in ACTA2 at 72 hr postinfection. CONCLUSIONS: Prostatic inflammation, even at the early stages, promotes atrophic and proliferative changes, and the upregulation of ErbB receptors together with dedifferentiation of smooth muscle cells. These data suggest that repetitive reinfections could lead to uncontrolled growth in the prostate gland.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter.

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.

Determination of cell proliferation using Mcm2 antigen and evaluation of apoptosis and TGF-beta1 expression in GH-secreting or clinically nonfunctioning pituitary adenomas.

Pituitary adenomas (PA) occasionally show aggressive behavior, with invasion of the surrounding tissues. The identification of markers able to recognize aggressive PA in early stages remains a challenge. We aimed to determine the expression of a new cell proliferation marker, Mcm2, and the presence of apoptosis in PA, and to evaluate the association of clinicopathological features with the apoptotic and proliferative indices. Additionally, the TGF-beta1 expression, an inducer of apoptosis, was determined. The proliferative index was determined in GH-secreting or clinically nonfunctioning PA using immunohistochemical (IH) methods for Mcm2 and Ki-67 antigens. The apoptosis was assessed by the TUNEL method and the TGF-beta1 expression by IH. A significant positive correlation was found between log Mcm2 index and log Ki-67 index (p < 0.001). Mcm2 and Ki-67 detected a similar number of proliferating cells. Mcm2 index showed a significant association with tumor extension (p = 0.02), but not with tumor invasion. Apoptosis was detected in 17% of the adenomas, with a maximum apoptotic index of 0.77%. Immunoreactivity to TGF-beta1 was observed in 77% of the adenomas, showing an association with tumor extension. We concluded that, in this sample, Mcm2 was similar to Ki-67 in the identification of the proliferating cells and that apoptosis was rare.

The effect of overweight and obesity on proliferation and activation of AKT and ERK in human endometria.

OBJECTIVE: To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women. METHODS: Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subject's body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays. RESULTS: In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subject's BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology. CONCLUSION: These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women.

Changes in the expression of insulin signaling pathway molecules in endometria from polycystic ovary syndrome women with or without hyperinsulinemia.

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder associated with insulin resistance and compensatory hyperinsulinemia. Scarce information is available on the expression of molecules involved in the insulin pathway in endometria from women with PCOS. Therefore, we examined the protein levels of insulin-signaling molecules, like insulin receptor, insulin-receptor substrate (IRS)-1, pIRS-1Y612, Akt, AS160, pAS160T642 and GLUT4 in endometria from PCOS women with or without hyperinsulinemia. Protein levels were assessed by Western blot and immunohistochemistry in 21 proliferative-phase endometria from control women (CE = 7), normoinssulinemic PCOS women (PCOSE-NI = 7) and hyperinsulinemic PCOS women (PCOSE-HI = 7). The data show no differences in the expression of insulin receptor between all groups as assessed by Western blot; however, IRS-1 and pIRS-1Y612 were lower in PCOSE-HI than controls and PCOSE-NI (P < 0.05). AS160 was detected in all analyzed tissues with similar expression levels between groups. Importantly, PCOSE-HI exhibited lower levels of pAS160T642 (P < 0.05) and of GLUT4 (P < 0.05) compared with CE. The immunohistochemistry for insulin receptor, IRS-1, Akt, AS160 and GLUT4 showed epithelial and stromal localization; IRS-1 staining was lower in PCOSE-HI (P < 0.05). In conclusion, human endometrium has the machinery for glucose uptake mediated by insulin. The diminished expression of GLUT4, as well as the lower level of pIRS-1Y612 and pAS160T642 exhibited by PCOSE-HI, suggests a disruption in the translocation of vesicles with GLUT4 to the cell surface in these patients.