RESUMO
The secreted protein transforming growth factor-beta (TGFß) plays essential roles, ranging from cell growth regulation and cell differentiation in both normal and cancer cells. In melanoma, TGFß acts as a potent tumor suppressor in melanoma by blocking cell cycle progression and inducing apoptosis. In the present study, we found TGFß to regulate cancer stemness in melanoma through the Smad signaling pathway. We discovered that TGFß/Smad signaling inhibits melanosphere formation in multiple melanoma cell lines and reduces expression of the CD133+ cancer stem cell subpopulation in a Smad3-dependent manner. Using preclinical models of melanoma, we further showed that preventing Smad3/4 signaling, by means of CRISPR knockouts, promoted both tumorigenesis and lung metastasis in vivo. Collectively, our results define new functions for the TGFß/Smad signaling axis in melanoma stem-cell maintenance and open avenues for new therapeutic approaches to this disease.
RESUMO
BACKGROUND: Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFß) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion. METHODS: In this study, we aimed at elucidating the molecular mechanisms underlying TGFß-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays. RESULTS: We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFß in melanoma cells, defining LIF as a novel TGFß downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFß-mediated LIF expression is required for TGFß-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFß-mediated inhibition of cell migration. Moreover, we found that TGFß-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFß/LIF-mediated cell cycle arrest and TGFß-induced gene activation of several pro-apoptotic genes. CONCLUSIONS: Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFß to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator Inibidor de Leucemia/biossíntese , Melanoma/genética , Neoplasias Cutâneas/genética , Fator de Crescimento Transformador beta1/genética , Adulto , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Inibidor de Leucemia/genética , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Cultura Primária de Células , Ligação Proteica , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta1/metabolismo , Melanoma Maligno CutâneoRESUMO
Although myostatin functions primarily as a negative regulator of skeletal muscle growth and development, accumulating biological and epidemiological evidence indicates an important contributing role in liver disease. In this study, we demonstrate that myostatin suppresses the proliferation of mouse Hepa-1c1c7 murine-derived liver cells (50%; p < 0.001) in part by reducing the expression of the cyclins and cyclin-dependent kinases that elicit G1-S phase transition of the cell cycle (p < 0.001). Furthermore, real-time PCR-based quantification of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (Malat1), recently identified as a myostatin-responsive transcript in skeletal muscle, revealed a significant downregulation (25% and 50%, respectively; p < 0.05) in the livers of myostatin-treated mice and liver cells. The importance of Malat1 in liver cell proliferation was confirmed via arrested liver cell proliferation (p < 0.05) in response to partial Malat1 siRNA-mediated knockdown. Myostatin also significantly blunted insulin-stimulated glucose uptake and Akt phosphorylation in liver cells while increasing the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS), a protein that is essential for cancer cell proliferation and insulin-stimulated glucose transport. Together, these findings reveal a plausible mechanism by which circulating myostatin contributes to the diminished regenerative capacity of the liver and diseases characterized by liver insulin resistance.
