Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

The thyroid status reprograms T cell lymphoma growth and modulates immune cell frequencies.

UNLABELLED: In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8(+) T cells, and higher percentage of CD19(+) B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas. KEY MESSAGES: T cell lymphoma phenotype is paradoxically influenced by thyroid status. Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination. Thyroid status affects the distribution of immune cell types in the tumor milieu. Thyroid status also modifies the nature of local and systemic immune responses.

Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance.

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

Thyroid status modulates T lymphoma growth via cell cycle regulatory proteins and angiogenesis.

We have shown in vitro that thyroid hormones (THs) regulate the balance between proliferation and apoptosis of T lymphoma cells. The effects of THs on tumor development have been studied, but the results are still controversial. Herein, we show the modulatory action of thyroid status on the in vivo growth of T lymphoma cells. For this purpose, euthyroid, hypothyroid, and hyperthyroid mice received inoculations of EL4 cells to allow the development of solid tumors. Tumors in the hyperthyroid animals exhibited a higher growth rate, as evidenced by the early appearance of palpable solid tumors and the increased tumor volume. These results are consistent with the rate of cell division determined by staining tumor cells with carboxyfluorescein succinimidyl ester. Additionally, hyperthyroid mice exhibited reduced survival. Hypothyroid mice did not differ significantly from the euthyroid controls with respect to these parameters. Additionally, only tumors from hyperthyroid animals had increased expression levels of proliferating cell nuclear antigen and active caspase 3. Differential expression of cell cycle regulatory proteins was also observed. The levels of cyclins D1 and D3 were augmented in the tumors of the hyperthyroid animals, whereas the cell cycle inhibitors p16/INK4A (CDKN2A) and p27/Kip1 (CDKN1B) and the tumor suppressor p53 (TRP53) were increased in hypothyroid mice. Intratumoral and peritumoral vasculogenesis was increased only in hyperthyroid mice. Therefore, we propose that the thyroid status modulates the in vivo growth of EL4 T lymphoma through the regulation of cyclin, cyclin-dependent kinase inhibitor, and tumor suppressor gene expression, as well as the stimulation of angiogenesis.

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase.

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.