Role of TGF-β signaling in the mechanisms of tamoxifen resistance

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling.     

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.     

Role of TGF-β signaling regulatory microRNAs in the pathogenesis of colorectal cancer

Transforming growth factor β (TGF-β) modulates tumor progression by regulating cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. Biological and pharmacological agonists/antagonists, the interplay between intracellular signaling pathways, and microRNAs (miRNAs) control the initiation and activation of the TGF-β signaling pathway. It has been proposed that the expression profiles of tumor suppressor and oncogenic TGF-β miRNAs may be used for the classification, diagnosis, and prognosis of human malignancies. Deregulated miRNAs and aberrant activation of TGF-β signaling are frequently found in human colorectal cancers (CRCs), but a little is known about their mechanisms of action in the development and progression of colorectal carcinoma. This review summarizes the current knowledge of the role of TGF-β signaling regulatory miRNAs in the pathogenesis of CRC for a better understanding and hence better management of this disease.     

Role of the transforming growth factor-β signaling pathway in the pathogenesis of colorectal cancer

The transforming growth factor-β (TGF-β) signaling pathway plays an important role in cancer cell proliferation, growth, metastasis, and apoptosis. It has been shown that TGF-β acts as a tumor suppressor in the early stages of the disease, and as a tumor promoter in its late stages. Mutations in the TGF-β signaling components, the TGF-β receptors and cytoplasmic signaling transducers, are frequently observed in colorectal carcinomas. Exploiting specific TGF-β receptor agonist and antagonist with antitumor properties may be a way of controlling cancer progression. This review summarizes the regulatory role of TGF-β signaling in the pathogenesis of colorectal cancer.     

Role of GSK-3β in the osteogenic differentiation of palatal mesenchyme

Introduction

The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.

Methods

Palates were harvested from GSK-3β, embryonic days 15.0–18.5 (e15.0–e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and −/− e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.

Results

GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β −/− palate cultures were “rescued” with the Wnt inhibitor, Dkk-1.

Conclusions

Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.     

Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.     

Role of STRAP in regulating GSK3β function and Notch3 stabilization

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.Key words: STRAP, GSK3β, Notch3, axin, lung cancer, ubiquitination     

Role of hippocalcin in mediating Aβ toxicity

Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-β (Aβ) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aβ plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aβ toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aβ and found them to be more susceptible to Aβ toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aβ treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.     

Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact.     

Role of Phosphoinositide 3-OH Kinase p110β in Skeletal Myogenesis

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit β (p110β) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110β delayed differentiation. We next generated mice with conditional deletion of p110β in skeletal muscle (p110β muscle knockout [p110β-mKO] mice). While young p110β-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110β-mKO mice were less glucose tolerant than old control mice. Overexpression of p110β accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110β had the opposite effect. p110β overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110β inhibition. These findings reveal a role for p110β during myogenesis and demonstrate that long-term reduction of skeletal muscle p110β impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.     

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.     

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.     

Inhibition of Wnt and PI3K Signaling Modulates GSK-3β Activity and Induces Morphological Changes in Cortical Neurons: Role of Tau Phosphorylation

Glycogen synthase kinase GSK-3β has been identified as one of the major candidates mediating tau hyperphosphorylation at the same sites as those present in tau protein in brain from Alzheimer′s disease (AD) patients. However, the signal transduction pathways involved in the abnormal activation of GSK-3β, have not been completely elucidated. GSK-3β activity is repressed by the canonical Wnt signaling pathway, but it is also modulated through the PI3K/Akt route. Recent studies have suggested that Wnt signaling might be involved in the pathophysiology of AD. On the other hand, modulators of the PI3K pathway might be reduced during aging leading to a sustained activation of GSK-3β, which in turn would increase the risk of tau hyperphosphorylation. The role of Wnt and PI3K signaling inhibition on the extent of tau phosphorylation and neuronal morphology has not been completely elucidated. Thus, in the present investigation we analyzed the effects of different negative modulators of the Wnt and the PI3K pathways on GSK-3β activation and phosphorylation of tau at the PHF-1 epitope in cortical cultured neurons and hippocampal slices from adult rat brain. Changes in the microtubule network were also studied. We found that a variety of Wnt and PI3K inhibitors, significantly increased tau phosphorylation at the PHF-1 site, induced the disarrangement of the microtubule network and the accumulation of tau within cell bodies. These changes correlated with alterations in neuronal morphology. Special issue article in honor of Dr. Ricardo Tapia.     

Role of phospholipase Cε in physiological phosphoinositide signaling networks

Receptor-initiated phospholipase C activation and generation of IP(3) and DAG are important common triggers for a diversity of signal transduction processes in many cell types. Contributing to this diversity is the existence and differential cellular and subcellular distribution of distinct phospholipase C isoforms with distinct regulatory properties. The recently identified PLCε enzyme is an isoform that is uniquely regulated by multiple upstream signals including ras-family GTP binding proteins as well as heterotrimeric G-proteins. In this review we will consider the well documented biochemical regulation of this isoform in the context of cell and whole animal physiology and in the context of other G protein-regulated PLC isoforms. These studies together reveal a surprisingly wide range of unexpected functions for PLCε in cellular signaling, physiology and disease.     

Nuclear GPCRs in cardiomyocytes: an insider's view of β-adrenergic receptor signaling

In recent years, we have come to appreciate the complexity of G protein-coupled receptor signaling in general and β-adrenergic receptor (β-AR) signaling in particular. Starting originally from three β-AR subtypes expressed in cardiomyocytes with relatively simple, linear signaling cascades, it is now clear that there are large receptor-based networks which provide a rich and diverse set of responses depending on their complement of signaling partners and the physiological state. More recently, it has become clear that subcellular localization of these signaling complexes also enriches the diversity of phenotypic outcomes. Here, we review our understanding of the signaling repertoire controlled by nuclear β-AR subtypes as well our understanding of the novel roles for G proteins themselves in the nucleus, with a special focus, where possible, on their effects in cardiomyocytes. Finally, we discuss the potential pathological implications of alterations in nuclear β-AR signaling.     

Essential Role of the p110β Subunit of Phosphoinositide 3-OH Kinase in Male Fertility

Phosphoinositide 3-kinases (PI3K) are key molecular players in male fertility. However, the specific roles of different p110 PI3K catalytic subunits within the spermatogenic lineage have not been characterized so far. Herein, we report that male mice expressing a catalytically inactive p110β develop testicular hypotrophy and impaired spermatogenesis, leading to a phenotype of oligo-azoospermia and defective fertility. The examination of testes from p110β-defective tubules demonstrates a widespread loss in spermatogenic cells, due to defective proliferation and survival of pre- and postmeiotic cells. In particular, p110β is crucially needed in c-Kit–mediated spermatogonial expansion, as c-Kit–positive cells are lost in the adult testis and activation of Akt by SCF is blocked by a p110β inhibitor. These data establish that activation of the p110β PI3K isoform by c-Kit is required during spermatogenesis, thus opening the way to new treatments for c-Kit positive testicular cancers.     

TGF-β family signaling in stem cells

Background

The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies.

Scope of review

The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells.

Major conclusions

Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells.

General significance

Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.