Estrogenic plant extracts reverse weight gain and fat accumulation without causing mammary gland or uterine proliferation.

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women.

Outgrowth of drug-resistant carcinomas expressing markers of tumor aggression after long-term TßRI/II kinase inhibition with LY2109761.

TGF-ß is produced excessively by many solid tumors and can drive malignant progression through multiple effects on the tumor cell and microenvironment. TGF-ß signaling pathway inhibitors have shown efficacy in preclinical models of metastatic cancer. Here, we investigated the effect of systemic LY2109761, a TGF-ß type I/II receptor (TßRI/TßRII) kinase inhibitor, in both a tumor allograft model and the mouse skin model of de novo chemically induced carcinogenesis in vivo. Systemic LY2109761 administration disrupted tumor vascular architecture and reduced myofibroblast differentiation of E4 skin carcinoma cells in a tumor allograft. In the 7,12-dimethyl-benzanthracene plus phorbol myristate acetate-induced skin chemical carcinogenesis model, acute dosing of established naive primary carcinomas with LY2109761 (100 mg/kg) every 8 hours for 10 days (100 mg/kg) diminished phospho-Smad2 (P-Smad2) levels and marginally decreased the expression of inflammatory and invasive markers. Sustained exposure to LY2109761 (100 mg/kg/d) throughout the tumor outgrowth phase had no effect on carcinoma latency or incidence. However, molecular analysis of resultant carcinomas by microarray gene expression, Western blotting, and immunohistochemistry suggests that long-term LY2109761 exposure leads to the outgrowth of carcinomas with elevated P-Smad2 levels that do not respond to drug. This is the first description of acquired resistance to a small-molecule inhibitor of the TßRI/TßRII kinase. Resultant carcinomas were more aggressive and inflammatory in nature, with delocalized E-cadherin and elevated expression of Il23a, laminin V, and matrix metalloproteinases. Therefore, TGF-ß inhibitors might be clinically useful for applications requiring acute administration, but long-term patient exposure to such drugs should be undertaken with caution.

Regulation of specific target genes and biological responses by estrogen receptor subtype agonists.

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERß. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by non-selectively triggering both ERα and ERß pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERß should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERß regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα-selective and ERß-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERß agonists are a promising new approach for treating specific conditions associated with menopause.

Estrogen receptor beta binds to and regulates three distinct classes of target genes.

Estrogen receptor beta (ERbeta) has potent antiproliferative and anti-inflammatory properties, suggesting that ERbeta-selective agonists might be a new class of therapeutic and chemopreventive agents. To understand how ERbeta regulates genes, we identified genes regulated by the unliganded and liganded forms of ERalpha and ERbeta in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ERalpha, whereas many genes were regulated by estradiol (E(2)). These results demonstrated that ERalpha requires a ligand to regulate a single class of genes. In contrast, ERbeta regulated three classes of genes. Class I genes were regulated primarily by unliganded ERbeta. Class II genes were regulated only with E(2), whereas class III genes were regulated by both unliganded ERbeta and E(2). There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ERbeta regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ERbeta binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFkappaB1, and SP1 sites were more enriched in class II genes. ERbeta bound to all three classes of genes, demonstrating that ERbeta binding is not responsible for differential regulation of genes by unliganded and liganded ERbeta. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ERbeta regulate three classes of genes by interacting with different transcription factors and coactivators.

Selective activation of estrogen receptor-beta target genes by 3,3'-diindolylmethane.

3,3'-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables that has antiproliferative and estrogenic activity. However, it is not clear whether the estrogenic effects are mediated through estrogen receptor (ER)alpha, ERbeta, or both ER subtypes. We investigated whether DIM has ER subtype selectivity on gene transcription. DIM stimulated ERbeta but not ERalpha activation of an estrogen response element upstream of the luciferase reporter gene. DIM also selectively activated multiple endogenous genes through ERbeta. DIM did not bind to ERbeta, indicating that it activates genes by a ligand-independent mechanism. DIM causes ERbeta to bind regulatory elements and recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER target genes, demonstrating that SRC-2 is required for transcriptional activation by DIM. Our results demonstrate that DIM is a new class of ERbeta-selective compounds, because it does not bind to ERbeta, but instead it selectively recruits ERbeta and coactivators to target genes.

Unliganded estrogen receptor-beta regulation of genes is inhibited by tamoxifen.

Tamoxifen can stimulate the growth of some breast tumors and others can become resistant to tamoxifen. We previously showed that unliganded ERbeta inhibits ERalpha-mediated proliferation of MCF-7 cells. We investigated if tamoxifen might have a potential negative effect on some breast cancer cells by blocking the effects of unliganded ERbeta on gene regulation. Gene expression profiles demonstrated that unliganded ERbeta upregulated 196 genes in MCF-7 cells. Tamoxifen significantly inhibited 73 of these genes by greater than 30%, including several growth-inhibitory genes. To explore the mechanism whereby unliganded ERbeta activates genes and how tamoxifen blocks this effect, we used doxycycline-inducible U2OS-ERbeta cells to produce unliganded ERbeta. Doxycycline produced a dose-dependent activation of the NKG2E, MSMB and TUB3A genes, which was abolished by tamoxifen. Unliganded ERbeta recruitment of SRC-2 to the NKG2E gene was blocked by tamoxifen. Our findings suggest that tamoxifen might exert a negative effect on ERbeta expressing tumors due to its antagonistic action on unliganded ERbeta.

Elevated cutaneous Smad activation associates with enhanced skin tumor susceptibility in organ transplant recipients.

PURPOSE: Nonmelanoma skin cancer incidence is enhanced >50-fold in patients taking antirejection drugs (ARD) following organ transplantation. Preclinical studies suggest that ARD treatment increases transforming growth factor-beta1 (TGF-beta1) levels, which contribute to enhanced tumor susceptibility independent of the immunosuppressive effects of ARDs. This study investigates whether TGF-beta signaling is elevated in transplant patients. EXPERIMENTAL DESIGN: Immunohistochemical tissue microarray analysis was used to determine the levels of TGF-beta1, TGF-beta2, TGF-beta3, TbetaRII, and activated P-Smad2/3 and P-Smad1/5/8, which are phosphorylated directly by distinct TGF-beta/BMP receptor complexes. We analyzed >200 cutaneous lesions and adjacent nonlesional skin samples from 87 organ transplant recipients, and 184 cutaneous lesions and adjacent skin samples from 184 individuals who had never received ARDs. RESULTS: We found significantly higher levels of P-Smad2 in both nonlesional and lesional tissue from transplant recipients compared with those not exposed to ARDs (P < or = 0.001). In contrast, P-Smad1/5/8, a marker of activation of the bone morphogenetic protein signaling pathway, was generally not expressed at higher levels in patients taking ARDs, including analysis of nonlesional skin, actinic keratoses, carcinoma in situ, or squamous cell carcinoma but was differentially expressed between keratoacanthoma from transplant recipients compared with those from non-transplant recipients (P < or = 0.005). CONCLUSIONS: Observation of elevated P-Smad2 levels in transplant recipients is consistent with the notion that elevated TGF-beta signaling may contribute to malignancy in organ transplant recipients. Disparate P-Smad1/5/8 expression levels between keratoacanthoma from the two patient groups might reflect the distinct BMP-responsive cell of origin for this hair follicle-derived lesion.

The type I TGF-beta receptor is covalently modified and regulated by sumoylation.

Post-translational sumoylation, the covalent attachment of a small ubiquitin-like modifier (SUMO), regulates the functions of proteins engaged in diverse processes. Often associated with nuclear and perinuclear proteins, such as transcription factors, it is not known whether SUMO can conjugate to cell-surface receptors for growth factors to regulate their functions. Here we show that the type I transforming growth factor-beta (TGF-beta) receptor, T beta RI, is sumoylated in response to TGF-beta and that its sumoylation requires the kinase activities of both T beta RI and the type II TGF-beta receptor, T beta RII. Sumoylation of T beta RI enhances receptor function by facilitating the recruitment and phosphorylation of Smad3, consequently regulating TGF-beta-induced transcription and growth inhibition. T beta RI sumoylation modulates the dissemination of transformed cells in a mouse model of T beta RI-stimulated metastasis. T beta RI sumoylation therefore controls responsiveness to TGF-beta, with implications for tumour progression. Sumoylation of cell-surface receptors may regulate other growth factor responses.

TGF beta inhibition for cancer therapy.

The importance of perturbation in transforming growth factor beta (TGFbeta) signaling for the onset and progression of cancer is well established. Many tumors over express TGFbeta, and high circulating levels of TGFbeta1 in cancer patients are frequently associated with poor prognosis. TGFbeta has context-dependent biphasic action during tumorigenesis. Because of this, it is essential to take due care about the selection of patients most likely to benefit from anti-TGFbeta therapy. Anti-TGFbeta therapy aims to target both the tumor cell and the tumor microenvironment and may well have systemic effects of relevance to tumorigenesis. Extra-tumoral targets include stromal fibroblasts, endothelial and pericyte cells during angiogenesis, and the local and systemic immune systems, all of which can contribute to the pro-oncogenic effects of TGFbeta. Many different approaches have been considered, such as interference with ligand synthesis using oligonucleotides, sequestration of extracellular ligand using naturally-occurring TGFbeta binding proteins, recombinant proteins or antibodies, targeting activation of latent TGFbeta at the cell surface, or signal transduction within the cell. Consideration of which patients might benefit most from anti-TGFbeta therapy should include not only tumor responses to TGFbeta (which depend on activation of other oncogenic pathways in the cancer cell), but also germline genetic variation between individuals. Ultimately, a deep understanding of the interacting networks of signal pathways that regulate TGFbeta outcome in tumor and host cells should allow judicial choice of drugs. This review discusses the progress made in the pre-clinical and clinical testing of TGFbeta inhibitors, and discusses considerations of target populations and potential drug regimens.

Genetic variants of Tgfb1 act as context-dependent modifiers of mouse skin tumor susceptibility.

The human TGFB1 gene is polymorphic, and genetic variants are associated with altered cancer risk. However, human genetic association studies have had variable outcomes because TGFbeta1 action is context-dependent. We used the murine skin model of chemical carcinogenesis in genetic linkage analysis of three independent Mus musculus NIH/Ola x (Mus spretus x M. musculus NIH/Ola)F1 backcrosses, to identify a skin tumor susceptibility locus, Skts14, on proximal chromosome 7. Tgfb1 maps at the peak of linkage. The mouse Tgfb1 gene is polymorphic, resulting in cis-regulated differential allelic mRNA expression between M. spretus and M. musculus in F1 mouse skin. This phenomenon is reflected in differential phospho-SMAD2 levels, downstream of TGFbeta signaling, between these two mouse species. In normal F1 mouse skin, the Tgfb1SPR allele is expressed at higher levels than the Tgfb1NIH allele, and this differential is accentuated by phorbol 12-myristate 13-acetate treatment. In benign F1 papillomas, this imbalance is reversed, possibly by selection against expression of a hyperactive Tgfb1SPR allele in TGFbeta growth-responsive tumors. We demonstrate that skin tumor susceptibility is altered by Tgfb1 gene dosage, but that manifestation of Tgfb1-linked skin tumor susceptibility in M. musculus NIH/Ola x (M. spretus x M. musculus NIH/Ola)F1 backcross mice depends on interactions with another unlinked tumor modifying locus, Skts15, that overlaps Tgfbm3 on chromosome 12. These findings illustrate the power of complex genetic interactions in determining disease outcome and have major implications to the assessment of disease risk in individuals harboring variant TGFB1 alleles.

Transforming growth factor-beta receptor inhibition enhances adenoviral infectability of carcinoma cells via up-regulation of Coxsackie and Adenovirus Receptor in conjunction with reversal of epithelial-mesenchymal transition.

Expression of the Coxsackie and Adenovirus Receptor (CAR) is frequently reduced in carcinomas, resulting in decreased susceptibility of such tumors to infection with therapeutic adenoviruses. Because CAR participates physiologically in the formation of tight-junction protein complexes, we examined whether molecular mechanisms known to down-regulate cell-cell adhesions cause loss of CAR expression. Transforming growth factor-beta (TGF-beta)-mediated epithelial-mesenchymal transition (EMT) is a phenomenon associated with tumor progression that is characterized by loss of epithelial-type cell-cell adhesion molecules (including E-cadherin and the tight junction protein ZO-1), gain of mesenchymal biochemical markers, such as fibronectin, and acquisition of a spindle cell phenotype. CAR expression is reduced in tumor cells that have undergone EMT in response to TGF-beta. This down-regulation results from repression of CAR gene transcription, whereas altered RNA stability and increased proteasomal protein degradation play no role. Loss of CAR expression in response to TGF-beta is accompanied by reduced susceptibility to adenovirus infection. Indeed, treatment of carcinoma cells with LY2109761, a specific pharmacologic inhibitor of TGF-beta receptor types I and II kinases, resulted in increased CAR RNA and protein levels as well as improved infectability with adenovirus. This was observed in cells induced to undergo EMT by addition of exogenous TGF-beta and in those that were transformed by endogenous autocrine/paracrine TGF-beta. These findings show down-regulation of CAR in the context of EMT and suggest that combination of therapeutic adenoviruses and TGF-beta receptor inhibitors could be an efficient anticancer strategy.