Angeles C. Tecalco-Cruz

Inhibitory Smad7: Emerging Roles in Health and Disease

Smad7 is an inhibitory Smad protein that blocks Transforming Growth Factor-beta (TGF-β) signaling through a negative feedback loop, also capable of mediating the crosstalk between TGF-β and other signaling pathways. Smad7 mRNA and protein levels are upregulated after TGF-β signaling; subsequently, Smad7 protein binds TGF-β type I receptor blocking R-Smad phosphorylation and eventually TGF-β signaling. Because of this inhibitory function, Smad7 can antagonize diverse cellular processes regulated by TGF-β such as cell proliferation, differentiation, apoptosis, adhesion and migration. Smad7 induction by different cytokines, besides TGF-β, is also critical for crosstalk/integration of a variety of signaling pathways, and relevant in the pathology of some diseases. Thus, Smad7 plays a key role in the control of various physiological events, and even in some pathological processes including fibrosis and cancer. This review highlights the main known functions of Smad7 with a particular focus on the relevance that alterations of Smad7 function may have in homeostasis, also describing some Smad7 emerging roles in the development of several human diseases that identify this protein as a potential therapeutic target.

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

Background: Human SKIL gene encodes for SNON, a negative regulator of the TGF-β/SMAD pathway. Results: We provide a molecular mechanism of transcriptional regulation of SKIL gene expression by TGF-β/SMADs. Conclusion: Transcriptional cofactor complex SNON-SMAD4 negatively controls the expression of SKIL gene. Significance: The formation and function of complex SNON-SMAD4 are impaired in cancer cells lacking SMAD4, which affects TGF β-target gene regulation. The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Mechanisms that Increase Stability of Estrogen Receptor Alpha in Breast Cancer.

Abstract Estrogen receptor alpha (ER) is a transcriptional regulator that controls the expression of genes related to cellular proliferation and differentiation in normal mammary tissue. However, the expression, abundance, and activity of this receptor are increased in 70% of breast cancers. The ER upregulation is facilitated by several molecular mechanisms, including protein stability, which represents an important strategy to maintain an active and functional repertoire of ER. Several proteins interact and protect ER from degradation by the ubiquitin‐proteasome system. Through diverse mechanisms, these proteins prevent polyubiquitination and degradation of ER, leading to an increase in ER protein levels; consequently, estrogen signaling and its physiologic effects are enhanced in breast cancer cells. Thus, increased protein stability seems to be one of the main reasons that ER is upregulated in breast cancer. Here, we highlight findings on the proteins and mechanisms that participate directly or indirectly in ER stability and their relevance to breast cancer.

Nucleo-cytoplasmic transport of estrogen receptor alpha in breast cancer cells

Approximately 70% cases of breast cancers exhibit high expression and activity levels of estrogen receptor alpha (ERα), a transcription regulator that induces the expression of genes associated with cellular proliferation and survival. These nuclear functions of the receptor are associated with the development of breast cancer. However, ERα localization is not static, but rather, dynamic with continuous shuttling between the nucleus and the cytoplasm. Interestingly, both the nuclear import and export of ERα are modulated by several stimuli that include estradiol, antiestrogens, and growth factors. As ERα nuclear accumulation is critical to the regulation of gene expression, nuclear export of this receptor modulates the intensity and duration of its transcriptional activity. Thus, the subcellular spatial distribution of ERα ensures tight modulation of its concentration in cellular compartments, as well as of its nuclear and extranuclear functions. In this review, we will discuss current findings regarding the biological importance of molecular mechanisms of, and proteins responsible for, the nuclear import and export of ERα in breast cancer cells.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

Tristetraprolin Represses Estrogen Receptor α Transactivation in Breast Cancer Cells

Background: Estrogen receptor α (ERα) mediates the effects of 17β-estradiol in mammary gland, and it is associated with the development of breast cancer tumors. Results: Tristetraprolin (TTP) represses ERα transactivation through its interaction with histone deacetylases. Conclusion:TTP acts as a novel ERα corepressor. Significance: TTP reduces estradiol-induced cell proliferation and tumor growth, suggesting it may be important in breast cancer development. Estrogen receptor α (ERα) mediates the effects of 17β-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer.

Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

Tristetraprolin (TTP) is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα), which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR). In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-β signals.

BACKGROUND SnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-β (TGF-β) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. METHODS We explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-β signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TβRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TβRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-β signaling was also studied. RESULTS SnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-β signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-β/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-β signaling. CONCLUSIONS ANS and PURO require TGF-β/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-β signaling. GENERAL SIGNIFICANCE Antibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-β signaling by controlling Ski and SnoN protein levels.

Polyubiquitination inhibition of estrogen receptor alpha and its implications in breast cancer

Estrogen receptor alpha (ERα) is detected in more than 70% of the cases of breast cancer. Nuclear activity of ERα, a transcriptional regulator, is linked to the development of mammary tumors, whereas the extranuclear activity of ERα is related to endocrine therapy resistance. ERα polyubiquitination is induced by the estradiol hormone, and also by selective estrogen receptor degraders, resulting in ERα degradation via the ubiquitin proteasome system. Moreover, polyubiquitination is related to the ERα transcription cycle, and some E3-ubiquitin ligases also function as coactivators for ERα. Several studies have demonstrated that ERα polyubiquitination is inhibited by multiple mechanisms that include posttranslational modifications, interactions with coregulators, and formation of specific protein complexes with ERα. These events are responsible for an increase in ERα protein levels and deregulation of its signaling in breast cancers. Thus, ERα polyubiquitination inhibition may be a key factor in the progression of breast cancer and resistance to endocrine therapy.

Differential expression and molecular interactions of chromosome region maintenance 1 and calreticulin exportins in breast cancer cells

Chromosome region maintenance 1 (CRM-1) and calreticulin (CALR) are two proteins that act as exportins for some nuclear receptors, in addition to other critical functions for cellular homeostasis. In several cancer types, CRM-1 and CALR are upregulated suggesting an imbalance in their functions. However, the regulation of CRM-1 and CALR, and their biological implications, are not completely known. Here, we evaluated the interplay between the levels of CRM-1 and CALR, and estrogen receptor alpha (ERα) status, in breast cancer cells. CRM-1 and CALR were upregulated in mammary tumors relative to normal mammary tissue. Furthermore, the mRNA and protein levels of CRM-1 and CALR were higher in breast cancer cells lacking ERα, in comparison with those that express ERα. Additionally, both proteins were distributed in the nucleus and cytoplasm in the two cell types. Importantly, we identified novel interactions for these exportins. First, we showed an interaction between CRM-1 and CALR, and then we identified that SUN1 and SUN2, two proteins localized in the nuclear envelop, were able to interact specifically with CRM-1, but not CALR. Interestingly, SUN1 and SUN2 expression seemed to be decreased in breast cancer, thereby affecting the interactions of these proteins with CRM-1, and possibly its actions as an exportin. Thus, our data suggest that expression levels for CRM-1 and CALR, the interaction between these exportins, and specific interactions of SUN1 and SUN2 with CRM-1 but not CALR, may be central elements in nucleo-cytoplasmic transport. Furthermore, deregulation of these elements may have serious implications in the progression of breast and other types of cancer.