Previous studies demonstrated that IGF-II binds directly to vitronectin (VN) while IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects including IGF binding protein-5 (IGFBP-5) production, IGF type-1 receptor autophosphorylation and cell migration. Thus, this study examined the hypothesis that a link between IGF-I and VN must occur and may be mediated through IGFBPs. Studies using competitive binding assays with VN and [125I]-labelled IGFs in the absence and presence of IGFBPs revealed IGFBP-4, IGFBP-5 and non-glycoyslated IGFBP-3 significantly enhance binding of IGF-I to VN, while IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogues indicate that glycosylation status of IGFBP-3 and the heparin-binding domains of IGFBP-3 and IGFBP-5 are important in this interaction. The functional significance of IGFs binding to VN on cell migration in MCF-7 breast carcinoma cells was examined and cell migration was found to be enhanced when VN was pre-bound to IGF-I in the presence of IGFBP-3, -4 and -5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding analogue, des(1- 3)IGF-I. Additionally, higher doses of IGFs in the presence of VN also could stimulate cell migration. Together, these data indicated the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells. Future directions for this work include investigations into the mechanisms underlying formation of the trimeric complex and the associated signalling pathways involved.

Insulin like Growth Factor Binding Proteins (IGFBPs) 1 to 6 have important physiological functions of regulating half life and bioavailability of Insulin like Growth Factors (IGFs). Consequently, these have been known to play important roles in embryonic development, postnatal growth and disease conditions like cancer. However, the physiological roles of IGFBPs are diverse and not restricted only to the IGF regulation. These molecules are found to be tumor suppressors or promoters depending on the physiological contexts. IGFBP-2 has been established as a tumor promoter and found to be unregulated in several cancers including breast, ovarian, prostate cancer and glioblastoma (GBM). Various in vitro and in vivo studies have convincingly demonstrated the role of IGFBP-2 in inducing tumor cell proliferation, migration, invasion and chemoresistance. Increased plasma and tissue levels of IGFBP-2 have been associated with poor clinical outcome with respect to patients’ response to the therapy, relapse and overall survival. Various studies so far have demonstrated the role of IGFBP-2 in promoting glioma cell proliferation, migration, invasion, chemoresistance and determining stamens of GICs (Glioma Initiating Cells). However, the exact mechanisms underlying these functions remain unknown. Apart from being a diagnostic and prognostic indicator, IGFBP-2 has also been proposed as a therapeutic target. Therefore it is essential to understand mechanistic insights into pro-tumorigenic functions of IGFBP-2. Apart from the conventional function of regulating IGFs, IGFBP-2 has been shown to have several IGF independent functions. In a previous study, we reported IGFBP-2 as an upstream regulator of β-catenin signaling pathway in breast cancer. Interestingly, this study linked the association of higher expression of IGFBP-2 and β-catenin with the lymph node metastasis status of breast cancer. β-catenin signaling has been considered as one of the most important pro-tumorigenic pathways in several cancers including glioblastoma. Considering the importance of IGFBP-2 and β-catenin signaling pathways in glioblastoma, it becomes important to evaluate regulation of β-catenin activity by IGFBP-2 in glioma and address its clinical relevance. With this aim, the objectives of this study are,  To study mechanism of IGFBP-2 mediated regulation of β-catenin signaling in glioma cells and prognostic significance of IGFBP-2 and β-catenin expression in GBM tissues.  Isolation of human single chain variable fragment (scFv) against IGFBP-2 and its characterization as an inhibitor for IGFBP-2 pro-tumorigenic functions. Towards this, we established stable IGFBP-2 knockdown U251 cell line and IGFBP-2 over expressing LN229 and U87 cell lines. IGFBP-2 modulation in these glioma cell lines did not alter the rate of proliferation but there was a significant effect on cellular migration and invasion. In case of U251 cell line, there was a significant decrease in the intracellular levels of β-catenin while in IGFBP-2 over expressing cell lines there was a marked increase in intracellular β-catenin suggesting that IGFBP-2 is involved in the regulation of β-catenin in these cells. It was observed that this regulation of β-catenin was not because of its transcriptional regulation or regulation of canonical Wnt ligands Wnt1, Wnt2 and Wnt3a. To further delineate the pathway and understand the mechanism behind regulation of β-catenin, upstream regulators of β-catenin were analyzed. GSK3β is an important negative regulator of β-catenin which primes it for ubiquitination and proteasomal degradation. Phosphorylation of GSK3β at Ser9 position renders this enzyme inactive. In our study, it was observed that there was a significant downregulation of p-GSK3β in U251 cells with IGFBP-2 knockdown and upregulation in IGFBP-2 over expressing cell lines. Overexpression of IGFBP-2 in LN229 and U87 cell lines resulted in considerable decrease in the GSK3β mediated phosphorylation of β-catenin. This study unequivocally established that regulation of β-catenin by IGFBP-2 is via inactivation of GSK3β. Furthermore, regulation of GSK3β was found to be due to action of FAK following binding of IGFBP-2 to integrins. The expression pattern of IGFBP-2 and β-catenin protein in the tumor tissues of 112 GBM patients was studied and its correlation with patient survival was analysed. In this analysis it was observed that co-expression of IGFBP-2 and β-catenin is a strong predictor of patient prognosis. These results further implied the importance of understanding IGFBP-2 and β-catenin association in GBM pathology. One of the interesting observations in our study is that, not only full length IGFBP-2 protein but also C-terminal domain of IGFBP-2 was sufficient to regulate β-catenin and other IGFBP-2 mediated functions. This strongly asserts the importance of C-terminal region of IGFBP-2 as a tumor promoter. Towards an attempt to develop an inhibitor for IGFBP-2 actions, we screened a human single chain variable fragment (scFv) library using phage display technique. From this screening, one scFv (B7J) was identified which was a binder of full length IGFBP-2 as well as C-terminal domain of IGFBP-2. This scFv showed inhibition of IGFBP-2-cell surface interaction and also efficiently inhibited IGFBP-2-induced signaling pathways like ERK, FAK and GSK3β/β-catenin. B7J treatment also neutralized regulation of IGFBP-2 transcriptional targets like MMP2 and CD24. Gelatin zymography indicated the ability of B7J to decrease matrix metalloprotease activity in the conditioned medium of glioma cells. These effects ultimately reflected on the IGFBP-2-induced cellular migratory and invasive behaviour which was largely abrogated by B7J scFv treatment. Considering the therapeutic importance of scFvs because of their small size, better tumor penetration and tumor retention capacity than full length antibody molecules, such kind of strategy could be of great importance in the management of GBM. Altogether, this study provides a mechanistic insight of IGFBP-2 mediated actions involving integrin/FAK/GSK3β/β-catenin pathways and the possible role of this crosstalk in the aggressiveness of glioblastoma. This study also provides a proof of principle that an inhibitor like anti IGFBP-2 scFv could be of importance for controlling invasive glioblastoma.

O tratamento do hipogonadismo hipogonadotrófico na mulher adulta com hipopituitarismo inclui diversas alternativas terapêuticas de estrógenos e progestágenos, sendo a via oral a de menor custo e a de maior comodidade à paciente. A rota estrogênica oral, entretanto, exerce marcada influência sobre o eixo hormônio de crescimento/fator de crescimento insulinasímile número 1 (GH/IGF-1) nessas mulheres. O tratamento com estrógenos orais, concomitante ao uso de GH em pacientes com hipopituitarismo, antagoniza as ações biológicas do GH e agrava as anormalidades de composição corporal e o metabolismo em geral. Presume-se que o estrógeno oral iniba a secreção/produção de IGF-1 através de um efeito de primeira passagem hepática, causando um aumento da secreção de GH através de inibição do feedback negativo de IGF-1 em mulheres normais. Isso é demonstrado clinicamente por redução da massa magra, aumento da massa gorda, perfil lipídico aterogênico e prejuízo do bem-estar psicológico. Alguns estudos apontam que os progestágenos com ação androgênica revertem o efeito de diminuição dos níveis séricos de IGF-1 induzida pelos estrógenos orais. Progestágenos neutros não apresentam esse efeito, porém, quanto maior a potência androgênica, maior será a reversão do efeito de diminuição de IGF-1. Na presente revisão da literatura, serão abordados os aspectos clínicos da reposição com estrógenos e progestágenos nas mulheres com hipopituitarismo, suas interações nas outras deficiências hormonais, bem como o impacto do uso de estrógenos sobre as ações metabólicas do GH. / Treatment of hypogonadotropic hypogonadism in adult woman with hypopituitarism can include a wide range of estrogen and progestogen treatment alternatives, and oral administration is the route of least cost and greatest patient comfort. The oral estrogen route has a major impact on the growth hormone-insulin-like growth factor I (GH-IGF-I) axis. Oral estrogen therapy, when given concurrently with GH to patients with hypopituitarism, antagonizes the biological effects of GH treatment and aggravates the abnormalities of body composition and the metabolism in general. It is presumed that oral estrogen suppresses the secretion/production of IGF-1 by a hepatic first-pass mechanism, resulting in increased GH secretion by means of suppressing the IGF-I negative feedback that is present in healthy women. This is manifest clinically in reduced lean body mass, increased fat mass, an atherogenic lipid profile and damage to psychological well-being. Some studies have indicated that progestogens with androgenic actions reverse the effect of reduced serum IGF-1 levels that is induced by the oral estrogens. Neutral progestogens do not exert this effect, however the stronger the androgenic potential, the more the effect of reduced IGF-1 will be reversed. This bibliographical review will deal with the clinical aspects of estrogen and progestogen replacement in women with hypopituitarism, their interactions with other hormone deficiencies and the impact of estrogen treatment on the metabolic actions of GH.

O tratamento do hipogonadismo hipogonadotrófico na mulher adulta com hipopituitarismo inclui diversas alternativas terapêuticas de estrógenos e progestágenos, sendo a via oral a de menor custo e a de maior comodidade à paciente. A rota estrogênica oral, entretanto, exerce marcada influência sobre o eixo hormônio de crescimento/fator de crescimento insulinasímile número 1 (GH/IGF-1) nessas mulheres. O tratamento com estrógenos orais, concomitante ao uso de GH em pacientes com hipopituitarismo, antagoniza as ações biológicas do GH e agrava as anormalidades de composição corporal e o metabolismo em geral. Presume-se que o estrógeno oral iniba a secreção/produção de IGF-1 através de um efeito de primeira passagem hepática, causando um aumento da secreção de GH através de inibição do feedback negativo de IGF-1 em mulheres normais. Isso é demonstrado clinicamente por redução da massa magra, aumento da massa gorda, perfil lipídico aterogênico e prejuízo do bem-estar psicológico. Alguns estudos apontam que os progestágenos com ação androgênica revertem o efeito de diminuição dos níveis séricos de IGF-1 induzida pelos estrógenos orais. Progestágenos neutros não apresentam esse efeito, porém, quanto maior a potência androgênica, maior será a reversão do efeito de diminuição de IGF-1. Na presente revisão da literatura, serão abordados os aspectos clínicos da reposição com estrógenos e progestágenos nas mulheres com hipopituitarismo, suas interações nas outras deficiências hormonais, bem como o impacto do uso de estrógenos sobre as ações metabólicas do GH. / Treatment of hypogonadotropic hypogonadism in adult woman with hypopituitarism can include a wide range of estrogen and progestogen treatment alternatives, and oral administration is the route of least cost and greatest patient comfort. The oral estrogen route has a major impact on the growth hormone-insulin-like growth factor I (GH-IGF-I) axis. Oral estrogen therapy, when given concurrently with GH to patients with hypopituitarism, antagonizes the biological effects of GH treatment and aggravates the abnormalities of body composition and the metabolism in general. It is presumed that oral estrogen suppresses the secretion/production of IGF-1 by a hepatic first-pass mechanism, resulting in increased GH secretion by means of suppressing the IGF-I negative feedback that is present in healthy women. This is manifest clinically in reduced lean body mass, increased fat mass, an atherogenic lipid profile and damage to psychological well-being. Some studies have indicated that progestogens with androgenic actions reverse the effect of reduced serum IGF-1 levels that is induced by the oral estrogens. Neutral progestogens do not exert this effect, however the stronger the androgenic potential, the more the effect of reduced IGF-1 will be reversed. This bibliographical review will deal with the clinical aspects of estrogen and progestogen replacement in women with hypopituitarism, their interactions with other hormone deficiencies and the impact of estrogen treatment on the metabolic actions of GH.

O tratamento do hipogonadismo hipogonadotrófico na mulher adulta com hipopituitarismo inclui diversas alternativas terapêuticas de estrógenos e progestágenos, sendo a via oral a de menor custo e a de maior comodidade à paciente. A rota estrogênica oral, entretanto, exerce marcada influência sobre o eixo hormônio de crescimento/fator de crescimento insulinasímile número 1 (GH/IGF-1) nessas mulheres. O tratamento com estrógenos orais, concomitante ao uso de GH em pacientes com hipopituitarismo, antagoniza as ações biológicas do GH e agrava as anormalidades de composição corporal e o metabolismo em geral. Presume-se que o estrógeno oral iniba a secreção/produção de IGF-1 através de um efeito de primeira passagem hepática, causando um aumento da secreção de GH através de inibição do feedback negativo de IGF-1 em mulheres normais. Isso é demonstrado clinicamente por redução da massa magra, aumento da massa gorda, perfil lipídico aterogênico e prejuízo do bem-estar psicológico. Alguns estudos apontam que os progestágenos com ação androgênica revertem o efeito de diminuição dos níveis séricos de IGF-1 induzida pelos estrógenos orais. Progestágenos neutros não apresentam esse efeito, porém, quanto maior a potência androgênica, maior será a reversão do efeito de diminuição de IGF-1. Na presente revisão da literatura, serão abordados os aspectos clínicos da reposição com estrógenos e progestágenos nas mulheres com hipopituitarismo, suas interações nas outras deficiências hormonais, bem como o impacto do uso de estrógenos sobre as ações metabólicas do GH. / Treatment of hypogonadotropic hypogonadism in adult woman with hypopituitarism can include a wide range of estrogen and progestogen treatment alternatives, and oral administration is the route of least cost and greatest patient comfort. The oral estrogen route has a major impact on the growth hormone-insulin-like growth factor I (GH-IGF-I) axis. Oral estrogen therapy, when given concurrently with GH to patients with hypopituitarism, antagonizes the biological effects of GH treatment and aggravates the abnormalities of body composition and the metabolism in general. It is presumed that oral estrogen suppresses the secretion/production of IGF-1 by a hepatic first-pass mechanism, resulting in increased GH secretion by means of suppressing the IGF-I negative feedback that is present in healthy women. This is manifest clinically in reduced lean body mass, increased fat mass, an atherogenic lipid profile and damage to psychological well-being. Some studies have indicated that progestogens with androgenic actions reverse the effect of reduced serum IGF-1 levels that is induced by the oral estrogens. Neutral progestogens do not exert this effect, however the stronger the androgenic potential, the more the effect of reduced IGF-1 will be reversed. This bibliographical review will deal with the clinical aspects of estrogen and progestogen replacement in women with hypopituitarism, their interactions with other hormone deficiencies and the impact of estrogen treatment on the metabolic actions of GH.

Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in circulation. IGFBPs 1-6 bind IGFs with high affinity and can either potentiate or inhibit IGF signaling in a context dependent manner. IGFBP2 is a 36 kDa protein and the second most abundant IGFBP in serum. Numerous studies in the recent past have implied a pro-tumorigenic role of IGFBP2. Elevated expression of IGFBP2 has been observed in multiple malignancies, including glioblastoma multiforme (GBM), ovarian, pancreatic, gastric, prostate, colon, breast, thyroid cancer and leukemia. In addition, increased expression of IGFBP2 in both tissues and serum of patients has been correlated with poor prognosis in prostate, glioblastoma and colon cancers. Pro-tumorigenic actions of IGFBP2 have been supported by in vitro studies, where IGFBP2 increases the tumorigenic potential of adrenocortical tumor cells, epidermoid carcinoma cells, glioma cells and ovarian cancer cells. Further, using xenograft animal models, the role of IGFBP2 in the progression of glioma has been established. In breast cancer, IGFBP2 was found to be over expressed in ductal carcinoma in situ and invasive breast cancer samples. IGFBP2 over expression has been shown to confer drug resistance and an increased expression has been reported to correlate with lymph node metastasis in T1 breast carcinomas. These reports implicate IGFBP2 in breast cancer biology. However, its role in breast cancer progression is not well defined. With this background, the following objectives were set for the current study: Functional characterization of IGFBP2 with respect to its possible role in breast cancer progression. Elucidation of the molecular mechanisms of IGFBP2 actions. Towards this, immunohistochemistry was performed on 132 invasive ductal carcinoma (IDC) grade III tumors using IGFBP2 specific antibody. It was observed that IGFBP2 expression was significantly higher in tumors in comparison to normal tissues that showed no detectable staining for IGFBP2. It was also observed that expression of IGFBP2 significantly correlated with the expression of ER. To understand the functional significance of IGFBP2 over expression in breast cancer, IGFBP2 was characterized with respect to proliferation, survival and tumor forming ability (in vitro and in vivo) in BT474 breast cancer cells. The knockdown of IGFBP2 expression resulted in suppression of colony formation (nearly 70%) in these breast cancer cells, which could be partially reversed upon exogenous addition of IGFBP2 protein. Proliferation assays using stable clones with knockdown of IGFBP2 in BT474 cells showed a significant decrease in proliferation as compared to vector transfected cells in the presence of serum. Culturing of IGFBP2 knockdown breast cancer cells in serum free medium resulted in their growth arrest in G0/G1 phase of cell cycle as compared to control cells, which progressed through the cell cycle. Prolonged culturing of IGFBP2 knockdown cells in serum free condition (up to 72 h) resulted in the increase of cells in sub G1 phase of the cell cycle. Prolonged depletion of growth factors (serum free conditions) could result in apoptosis of these G1 arrested IGFBP2 knockdown cells. When serum starved IGFBP2 knockdown cells were treated with IGFBP2 protein, the cells arrested in G0/G1 phase were able to progress through the cell cycle and concomitant decrease in sub G1 fraction was observed. Knockdown of IGFBP2 resulted in significantly decreased number and visibly smaller colonies in anchorage independent conditions in vitro. Consistent with this observation, in vivo tumor xenograft formation with IGFBP2 knockdown cells also showed significant reduction in tumor weight as compared to vector generated tumors. These results imply that IGFBP2 has potent growth promoting effects on breast cancer and acts as a mitogen/survival factor for breast cancer cells. To elucidate the molecular mechanisms underlying the pro-tumorigenic effects of IGFBP2, the transcriptome profile following IGFBP2 perturbation in breast cancer cells was determined. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several events, notably cell cycle, p53 and Wnt signaling, as revealed by Gene Set Enrichment Analysis (GSEA). To further validate these results in breast cancer tissues, whole genome expression analysis was performed in 19 breast tumor samples which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. In comparison to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK, focal adhesion and Wnt signaling pathway. In order to identify the genes commonly regulated by IGFBP2 in cell lines and tumors, the gene expression profiles of IGFBP2 positive versus IGFBP2 negative tumors and IGFBP2 knockdown breast cancer cells were compared. 347 genes were found to be common among IGFBP2 regulated genes in tumors and cell line. The most significant networks representing the web of interactions among these genes were found to be associated with cellular growth and proliferation, cellular movement and nucleic acid metabolism, indicating an association of IGFBP2 expression phenotype to the distinct changes in expression of genes associated with the regulation of cellular growth and migration. Silencing of IGFBP2 in BT474 cells resulted in a reduced IGF signaling as evidenced by the reduced phosphorylation of IGF1R and concomitantly that of ERK. This effect could be reversed upon addition of the IGFBP2 protein, implying that IGFBP2 potentiates IGF signaling in breast cancer cells. Besides IGF ligand and their receptors, regulation of proliferation associated genes like CENPF, TOP2A, CCND1 and FOXM1 by IGFBP2 was observed, thus providing a molecular basis for the pro-proliferative effects of IGFBP2 on breast cancer cells. Addition of IGFBP2 to immortal breast cells resulted in reduced IGF1R signaling and reduced pERK and pAKT signaling. Additionally, the genes involved in cellular proliferation were down regulated upon IGFBP2 treatment in immortal cells. IGFBP2 knockdown clones had reduced expression of FOXM1, a key regulator of cell cycle for G1/S and G2/M transition, and M phase progression. The regulation of CENPF and CCND1 genes was established following over expression of FOXM1 in IGFBP2 knockdown cells. One of the important and novel finding of this study is the regulation of Wnt signaling pathway genes such as CCND1, MMP7, FGF18, MYCBP, FN1 and survivin by IGFBP2. In support of this, β-catenin protein was found to be regulated by IGFBP2 in breast cancer and GBM cells, as evidenced by knockdown and over expression studies. Furthermore, regulation of β-catenin by IGFBP2 was found to involve integrin-FAK and IGF1R signaling. Another important finding of this study is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. When expression of both IGFBP2 and β-catenin was correlated with the lymph node status of breast cancers, a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis was observed in comparison with tumors that did not show staining for either protein. Altogether, in this study employing genomic, cellular and molecular approaches, a pro- tumorigenic role for IGFBP2 in breast cancer has been established. Furthermore, this study provides novel insights into the molecular mechanisms employed by IGFBP2 involving IGF1R, FAK and Wnt signaling pathways during breast cancer progression.