Thyroid hormones (TH) play a fundamental role in diverse processes, including cellular movement. Cell migration requires the integration of events that induce changes in cell structure towards the direction of migration. These actions are driven by actin remodeling and stabilized by the development of adhesion sites to extracellular matrix via transmembrane receptors linked to the actin cytoskeleton. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that promotes cell migration and invasion through the control of focal adhesion turnover. In this work, we demonstrate that the thyroid hormone triiodothyronine (T3) regulates actin remodeling and cell movement in breast cancer T-47D cells through the recruitment of FAK. T3 controls FAK phosphorylation and translocation at sites where focal adhesion complexes are assembled. This process is triggered via rapid signaling to integrin αV/β3, Src, phosphatidylinositol 3-OH kinase (PI3K), and FAK. In addition, we established a cellular model with different concentration of T3 levels: normal, absence, and excess in T-47D breast cancer cells. We found that the expression of Src, FAK, and PI3K remained at normal levels in the excess of T3 model, while it was significantly reduced in the absence model. In conclusion, these results suggest a novel role for T3 as an important modulator of cell migration, providing a starting point for the development of new therapeutic strategies for breast cancer treatment.
The thyroid hormone triiodothyronine (T3) plays a fundamental role in growth regulation, differentiation, metabolism and cellular movement. These processes are particularly important considering that deregulation of T3 levels could promote abnormal responsiveness of mammary epithelial cells, which may lead to the development and progression of breast cancer (BC). Once cells migrate and invade different tissues, BC metastasis is the main cause of cancer-related death because it is particularly difficult to revert this multistep process. Cell migration integrates several steps that induce changes in cell structure and morphology to promote BC cell invasion. These sequential steps include actin cytoskeleton remodeling, focal adhesion complex formation and, finally, the turnover of branched actin filament networks. In this article, we demonstrate that T3 has the ability to modify the Epithelial-Mesenchymal Transition process. In addition, we show that T3 induces actin cytoskeleton reorganization, triggers focal adhesion formation and, as a consequence, promotes actin nucleation via non-genomic pathway. These events are specifically modulated by T3 via integrin αvβ3 to FAK/paxillin/cortactin/N-WASP/Arp2/3 complex signaling pathway, increasing cell adhesion, migration and invasion of T-47D BC cells. We suggest that T3 influences the progression of tumor metastasis by controlling signaling pathways that converge in cell motility. This knowledge is crucial for the development of novel therapeutic strategies for BC treatment.
Background: Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17β-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. Objectives: The aim of this work was to evaluate the molecular switch Cdk5 kinase/protein phosphatase 2A (PP2A) in the control of WAVE1 protein (phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. Results: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A dominant negative construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. Conclusion: These results suggest that E2 and P4 dynamically regulate neuron morphology through nongenomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.
Breast cancer (BC) is a major public health problem affecting women worldwide. Approximately 80% of diagnosed cases are hormone-dependent breast cancers. These hormones are known to stimulate tumor development and progression. In this setting, tentative evidence suggests that luteinizing hormone (LH) may also play a role in tumors. In BC cells that express functional LH receptors (LHR), this hormone regulates cell migration and invasion by controlling several kinases that activate actin cytoskeletal proteins. In this article, we show that LH induces phosphorylation of paxillin and its translocation toward the plasmatic membrane, where focal adhesion complexes are assembled. This process is triggered via a rapid extra-gonadal LHR signaling to Src/FAK/paxillin, which results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-WASP. As a consequence, Arp2/3 complexes induce actin polymerization, essential to promote cell adhesion, migration, and invasion, thus enhancing metastatic spread of tumoral cells. Our findings provide relevant information about how gonadotrophins exert their action in BC. This information helps us understand the extragonadal effects of LH on BC metastasis. It may provide new perspectives for therapeutic treatment, especially for women with high serum levels of gonadotrophins.
Purpose of the study: To evaluate the role of obesity in tumor progression and tumor microenvironment (TME) dynamic after the inhibition of nitric oxide synthase (NOS) in syngeneic murine models engrafted with triple negative breast cancer (TNBC) tumors. For this study, we wanted to deepen the results of our recently completed phase I/II clinical trial, where treatment with NG-methyl-L-arginine (L-NMMA, a pan-NOS inhibitor) increased the response rate to chemotherapy (docetaxel) in obese metastatic TNBC (ER-/PR-/HER2-) patients to 86% vs. 60% in normal weight individuals. TME analysis from responders revealed a neutrophil phenotype shift from protumor N2 to antitumor N1. We thus hypothesize that combined treatment with L-NMMA and docetaxel enhances antitumor effect by modulating TME in obese mice with TNBC. Experimental procedures: 3-week old female C57BL/6 mice were fed for 10 weeks either with high fat diet (HFD) or normal diet (ND). At week 13, TNBC mouse E0771 tumor cells were injected into the mice mammary fat pad. Once tumors reached 100 mm3, the animals were randomized to four groups: vehicle; docetaxel; L-NMMA; and docetaxel plus L-NMMA treatment. Tumor volume was measured throughout the experiment and growth rate was compared between groups at the end of treatment. Blood, tumor and perigonadal white adipose tissue (pgWAT) were collected. Cytokines and nitrite/nitrate levels (NO2-/NO3-, a NO indicator) were measured from blood and conditioned media (CM) from pgWAT using appropriate kits. Tumor immune cell infiltration was evaluated by immunohistochemistry. GraphPad software was used for statistical analysis and P-values <0.05 were considered to describe statistically significant differences between groups. Results: Tumor growth rate was significantly higher in HFD mice compared to ND mice. Treatment with the docetaxel/L-NMMA combination significantly slowed tumor growth in HFD mice (p=0.015), and showed a similar trend in ND mice (p=0.92) vs. the corresponding vehicle-treated groups. The reduction of tumor growth was significantly higher in HFD mice vs. ND mice (median of differences -2.0, p=0.031). HFD mice presented higher levels of pro-inflammatory cytokines, [IL-1b (p=0.011), IL-6 (p<0.0001) and TNFa (p<0.0001)] vs ND mice in pgWAT CM, as well as increased levels of NO2-/NO3- and expression of iNOS in tumors and in perigonadal adipose tissue. Conclusion: HFD mice had a pro-inflammatory profile with a significantly faster tumor growth and higher expression of iNOS in tumor and adipose tissues. Treatment with the docetaxel/L-NMMA combination resulted in a more significant anti-tumor effect in HFD mice, likely through remodeling of TME. Spatial analysis, including CODEX and immunophenotyping are being conducted to determine the TME influence in obesity associated TNBC and the effect of NOS inhibition. Citation Format: Ivonne Uzair, Kai Sun, Ann Anselme, Wei Qian, Jianying Zhou, Roberto Rosato, Jenny Chang. NOS inhibition enhances docetaxel-mediated anti-tumor effect in obese mice with triple negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3991.
All-trans retinoic acid (RA) is the primary metabolite of vitamin A and controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, known as retinoids, are promising agents in treating and chemoprevention different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with the metastatic behavior of tumors. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that RA plus FAKi (RA+FAKi) approach could improve inhibition of tumor progression. By in silico analysis, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. In metastatic BC cells, we reveal that genes encoding proteins that FAK directly or indirectly modulates are deregulated compared to normal cells. We showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. We also evidenced that RA effects are extrapolated to other cancer cells, including the human cervical carcinoma cells HeLa. In an orthotopic assay of LM3 tumor growth, RA and FAKi administered separately reduce tumor growth; however, the combined treatment induces the more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduces metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance would reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbated these effects suggesting that the sensibility to RA therapies could be increased with FAKi coadministration in BC tumors.
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