Contribution of endoplasmic reticulum stress, MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells.

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

Integrin αvß3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells.

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvß3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvß3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvß3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvß3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Thyroid hormones and their membrane receptors as therapeutic targets for T cell lymphomas.

Thyroid hormones (THs) are important regulators of metabolism, differentiation and cell proliferation. They can modify the physiology of human and murine T cell lymphomas (TCL). These effects involve genomic mechanisms, mediated by specific nuclear receptors (TR), as well as nongenomic mechanisms, that lead to the activation of different signaling pathways through the activation of a membrane receptor, the integrin αvß3. Therefore, THs are able to induce the survival and growth of TCL. Specifically, the signaling induced by THs through the integrin αvß3 activates proliferative and angiogenic programs, mediated by the regulation of the vascular endothelial growth factor (VEGF). The genomic or pharmacologic inhibition of integrin αvß3 reduces the production of VEGF and induces cell death both in vitro and in xenograft models of human TCL. Here we review the mechanisms involved in the modulation of the physiology of TCL induced by THs, the analysis of the interaction between genomic and nongenomic actions of THs and their contribution to T cell lymphomagenesis. These actions of THs suggest a novel mechanism for the endocrine modulation of the physiopathology of TCL and they provide a potential molecular target for its treatment.

Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer.

BACKGROUND: The biological relevance of nuclear ErbB-2/HER2 (NuclErbB-2) presence in breast tumors remains unexplored. In this study we assessed the clinical significance of ErbB-2 nuclear localization in primary invasive breast cancer. The reporting recommendations for tumor marker prognostic studies (REMARK) guidelines were used as reference. METHODS: Tissue microarrays from a cohort of 273 primary invasive breast carcinomas from women living in Chile, a Latin American country, were examined for membrane (MembErbB-2) and NuclErbB-2 expression by an immunofluorescence (IF) protocol we developed. ErbB-2 expression was also evaluated by immunohistochemistry (IHC) with a series of antibodies. Correlation between NuclErbB-2 and MembErbB-2, and between NuclErbB-2 and clinicopathological characteristics of tumors was studied. The prognostic value of NuclErbB-2 in overall survival (OS) was evaluated using Kaplan-Meier method, and Cox model was used to explore NuclErbB-2 as independent prognostic factor for OS. RESULTS: The IF protocol we developed showed significantly higher sensitivity for detection of NuclErbB-2 than IHC procedures, while its specificity and sensitivity to detect MembErbB-2 were comparable to those of IHC procedures. We found 33.6% NuclErbB-2 positivity, 14.2% MembErbB-2 overexpression by IF, and 13.0% MembErbB-2 prevalence by IHC in our cohort. We identified NuclErbB-2 positivity as a significant independent predictor of worse OS in patients with MembErbB-2 overexpression. NuclErbB-2 was also a biomarker of lower OS in tumors that overexpress MembErbB-2 and lack steroid hormone receptors. CONCLUSIONS: We revealed a novel role for NuclErbB-2 as an independent prognostic factor of poor clinical outcome in MembErbB-2-positive breast tumors. Our work indicates that patients presenting NuclErbB-2 may need new therapeutic strategies involving specific blockage of ErbB-2 nuclear migration.

Inhibition of Integrin αVß3 Signaling Improves the Antineoplastic Effect of Bexarotene in Cutaneous T-Cell Lymphoma.

Bexarotene is a specific retinoid X receptor agonist that has been used for the treatment of cutaneous T-cell lymphoma (CTCL). Because bexarotene causes hypothyroidism, it requires the administration of levothyroxine. However, levothyroxine, in addition to its ubiquitous nuclear receptors, can activate the αVß3 integrin that is overexpressed in CTCL, potentially interfering the antineoplastic effect of bexarotene. We thus investigated the biological effect of levothyroxine in relation to bexarotene treatment. Although in isolated CTCL cells levothyroxine decreased, in an αVß3-dependent manner, the antineoplastic effect of bexarotene, levothyroxine supplementation in preclinical models was necessary to avoid suppression of lymphoma immunity. Accordingly, selective genetic and pharmacologic inhibition of integrin αVß3 improved the antineoplastic effect of bexarotene plus levothyroxine replacement while maintaining lymphoma immunity. Our results provide a mechanistic rationale for clinical testing of integrin αVß3 inhibitors as part of CTCL regimens based on bexarotene administration. TEASER: Inhibiting αVß3 integrin improves the antineoplastic effect of bexarotene while maintaining lymphoma immunity.

Thyroid status regulates the tumor microenvironment delineating breast cancer fate.

The patient's hormonal context plays a crucial role in the outcome of cancer. However, the association between thyroid disease and breast cancer risk remains unclear. We evaluated the effect of thyroid status on breast cancer growth and dissemination in an immunocompetent mouse model. For this, hyperthyroid and hypothyroid Balb/c mice were orthotopically inoculated with triple-negative breast cancer 4T1 cells. Tumors from hyperthyroid mice showed an increased growth rate and an immunosuppressive tumor microenvironment, characterized by increased IL-10 levels and decreased percentage of activated cytotoxic T cells. On the other hand, delayed tumor growth in hypothyroid animals was associated with increased tumor infiltration of activated CD8+ cells and a high IFNγ/IL-10 ratio. Paradoxically, hypothyroid mice developed a higher number of lung metastasis than hyperthyroid animals. This was related to an increased secretion of tumor CCL2 and an immunosuppressive systemic environment, with increased proportion of regulatory T cells and IL-10 levels in spleens. A lower number of lung metastasis in hyperthyroid mice was related to the reduced presence of mesenchymal stem cells in tumors and metastatic sites. These animals also exhibited decreased percentages of regulatory T lymphocytes and myeloid-derived suppressor cells in spleens but increased activated CD8+ cells and the IFNγ/IL-10 ratio. Therefore, thyroid hormones modulate the cellular and cytokine content of the breast tumor microenvironment. A better understanding of the mechanisms involved in these effects could be a starting point for the discovery of new therapeutic targets for breast cancer.

A Penicillin Derivative Exerts an Anti-Metastatic Activity in Melanoma Cells Through the Downregulation of Integrin αvß3 and Wnt/ß-Catenin Pathway.

The synthetic triazolylpeptidyl penicillin derivative, named TAP7f, has been previously characterized as an effective antitumor agent in vitro and in vivo against B16-F0 melanoma cells. In this study, we investigated the anti-metastatic potential of this compound on highly metastatic murine B16-F10 and human A375 melanoma cells. We found that TAP7f inhibited cell adhesion, migration and invasion in a dose-dependent manner. Additionally, we demonstrated that TAP7f downregulated integrin αvß3 expression and Wnt/ß-catenin pathway, a signaling cascade commonly related to tumor invasion and metastasis. Thus, TAP7f reduced both the enzymatic activity and the expression levels of matrix-metalloproteinases-2 and -9 in a time dependent manner. Moreover, TAP7f inhibited the expression of the transcription factor Snail and the mesenchymal markers vimentin, and N-cadherin, and up-regulated the expression of the epithelial marker E-cadherin, suggesting that the penicillin derivative affects epithelial-mesenchymal transition. Results obtained in vitro were supported by those obtained in a B16-F10-bearing mice metastatic model, that showed a significant TAP7f inhibition of lung metastasis. These findings suggest the potential of TAP7f as a chemotherapeutic agent for the treatment of metastatic melanoma.

Non-genomic Actions of Thyroid Hormones Regulate the Growth and Angiogenesis of T Cell Lymphomas.

T-cell lymphomas (TCL) are a heterogeneous group of aggressive clinical lymphoproliferative disorders with considerable clinical, morphological, immunophenotypic, and genetic variation, including ~10-15% of all lymphoid neoplasms. Several evidences indicate an important role of the non-neoplastic microenvironment in promoting both tumor growth and dissemination in T cell malignancies. Thus, dysregulation of integrin expression and activity is associated with TCL survival and proliferation. We found that thyroid hormones acting via the integrin αvß3 receptor are crucial factors in tumor microenvironment (TME) affecting the pathophysiology of TCL cells. Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and induced and an angiogenic program via the up-regulation of the vascular endothelial growth factor (VEGF). This was observed both on different TCL cell lines representing the different subtypes of human hematological malignancy, and in preclinical models of TCL tumors xenotransplanted in immunodeficient mice as well. Moreover, development of solid tumors by inoculation of murine TCLs in syngeneic hyperthyroid mice, showed increased tumor growth along with increased expression of cell cycle regulators. The genomic or pharmacological inhibition of integrin αvß3 decreased VEGF production, induced TCL cell death and decreased in vivo tumor growth and angiogenesis. Here, we review the non-genomic actions of THs on TCL regulation and their contribution to TCL development and evolution. These actions not only provide novel new insights on the endocrine modulation of TCL, but also provide a potential molecular target for its treatment.

Hypothyroidism-related zinc deficiency leads to suppression of T lymphocyte activity.

PURPOSE: Hypothyroidism has been shown to induce immunosuppression and both the thyroid status and immunity are affected by zinc deficiency. However, the impact of hypothyroidism on zinc metabolism and its possible relationship with the immune status has not yet been deeply explored. Here, our aim was to study whether hypothyroidism may alter zinc metabolism and thus lead to the impairment of T lymphocyte activity. METHODS: Variations in the distribution of zinc in the body were evaluated in PTU-treated hypothyroid mice. The effects of hypothyroidism and zinc deficiency were studied on T lymphocyte proliferation after stimulation both in vitro and in vivo. For in vitro assays, thyroid hormone-free or zinc chelator (TPEN or DTPA)-supplemented media were used. For in vivo assays, lymphocyte activity was evaluated in cells from hypothyroid, T3-treated, and zinc-supplemented mice. RESULTS: Hypothyroid mice showed lower levels of zinc in femur and lymph nodes than controls. T3 and zinc supplementation reversed these effects. In vitro, both thyroid hormone and zinc deficiency led to a decreased response to mitogen stimulation. However, only zinc deficiency was able to induce lymphocyte apoptosis. Mitogen-stimulated T cells from hypothyroid mice showed impaired proliferation, accompanied by decreased activation of PKC and lower levels of p-ERK, effects that were reversed by T3 replacement or zinc supplementation. CONCLUSIONS: Our results show an important role of zinc deficiency in hypothyroid-mediated T-cell suppression and suggest the importance of evaluating zinc levels and restoring them when necessary to maintain an efficient immune response in hypothyroid patients.

THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors.

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients.

Novel role of signal transducer and activator of transcription 3 as a progesterone receptor coactivator in breast cancer.

Interactions between progesterone receptor (PR) and signal transducer and activator of transcription 3 (Stat3)-mediated signaling pathways have already been described. In the present study, we explored the capacity of Stat3 to functionally interact with progesterone receptor (PR) and modulate PR transcriptional activation in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) induced the association of a PR/Stat3 complex in which Stat3 acts as a coactivator of PR. We demonstrated that Stat3 activation is required for MPA modulation of the endogenous genes bcl-X and p21(CIP1) which are involved in MPA-induced cell cycle regulation. Stat3 activity as a coactivator of PR was observed in both the classical and nonclassical ligand activated-PR transcriptional mechanisms, since the effects described were identified in the bcl-X promoter which contains a progesterone responsive element and in the p21(CIP1) promoter which carries Sp1 binding sites where PR is recruited via the transcription factor Sp1. The data herein presented identifies a potential therapeutic intervention for PR-positive breast tumors consisting of targeting Stat3 function or PR/Stat3 interaction which will result in the inhibition of PR function.

Progesterone receptor induces ErbB-2 nuclear translocation to promote breast cancer growth via a novel transcriptional effect: ErbB-2 function as a coactivator of Stat3.

Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.