RESUMO
OBJECTIVE: The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS: A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS: The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS: The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.
Assuntos
Anticorpos Antineoplásicos/química , Proteínas de Homeodomínio/análise , Neoplasias Ovarianas/química , Animais , Anticorpos Antineoplásicos/imunologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Feminino , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transplante HeterólogoRESUMO
Nucleotide excision repair (NER) is a key pathway for removing DNA damage that destabilizes the DNA double helix. During NER a protein complex coordinates to cleave the damaged DNA strand on both sides of the damage. The resulting lesion-containing oligonucleotide is displaced from the DNA and a replacement strand is synthesized using the undamaged strand as template. Ultraviolet (UV) light is known to induce two primary forms of DNA damage, the cyclobutane pyrimidine dimer and the 6-4 photoproduct, both of which destabilize the DNA double helix. The uvs9 strain of Chlamydomonas reinhardtii was isolated based on its sensitivity to UV light and was subsequently shown to have a defect in NER. In this work, the UVS9 gene was cloned through molecular mapping and shown to encode a homolog of XPG, the structure-specific nuclease responsible for cleaving damaged DNA strands 3' to sites of damage during NER. 3' RACE revealed that the UVS9 transcript is alternatively polyadenylated. The predicted UVS9 protein is nearly twice as long as other XPG homologs, primarily due to an unusually long spacer region. Despite this difference, amino acid sequence alignment of UVS9p with XPG homologs revealed a new conserved domain involved in TFIIH interaction.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Endonucleases/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , DNA de Plantas/metabolismo , DNA de Plantas/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Genes de Plantas , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro , Alinhamento de Sequência , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
An association between lymph node metastasis and poor prognosis in breast cancer was observed decades ago. However, the mechanisms by which tumor cells infiltrate the lymphatic system are not completely understood. Recently, it has been proposed that the lymphatic system has an active role in metastatic dissemination and that tumor-secreted growth factors stimulate lymphangiogenesis. We therefore investigated whether SIX1, a homeodomain-containing transcription factor previously associated in breast cancer with lymph node positivity, was involved in lymphangiogenesis and lymphatic metastasis. In a model in which human breast cancer cells were injected into immune-compromised mice, we found that SIX1 expression promoted peritumoral and intratumoral lymphangiogenesis, lymphatic invasion, and distant metastasis of breast cancer cells. SIX1 induced transcription of the prolymphangiogenic factor VEGF-C, and this was required for lymphangiogenesis and lymphatic metastasis. Using a mouse mammary carcinoma model, we found that VEGF-C was not sufficient to mediate all the metastatic effects of SIX1, indicating that SIX1 acts through additional, VEGF-C-independent pathways. Finally, we verified the clinical significance of this prometastatic SIX1/VEGF-C axis by demonstrating coexpression of SIX1 and VEGF-C in human breast cancer. These data define a critical role for SIX1 in lymphatic dissemination of breast cancer cells, providing a direct mechanistic explanation for how VEGF-C expression is upregulated in breast cancer, resulting in lymphangiogenesis and metastasis.