RESUMO
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. METHODS: The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. RESULTS: PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. CONCLUSIONS: Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC.
Assuntos
Proteínas Oncogênicas/genética , Proteína Quinase C/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA , Modelos Animais de Doenças , Feminino , Expressão Gênica , Xenoenxertos , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Família Multigênica/genética , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Proteínas Oncogênicas/metabolismo , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Transforming growth factor ß (TGF-ß) signaling plays an integral role in skeletal development. Conditional deletion of the TGF-ß type II receptor (Tgfbr2) from type II Collagen (Col2a) expressing cells results in defects in development of the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously used microarray analysis to search for marker genes of AF as well as transcription factors regulated by TGF-ß during AF development. The transcription factor avian erythroblastosis virus E-26 (v-ets) oncogene related (Erg) was identified in the microarray screen as a candidate regulator of AF development. To study the effects of TGF-ß on AF differentiation and the role of Erg in this process, we used mouse sclerotome grown in micromass cultures. At 0.5ng TGF-ß/ml, sclerotome cells started to express markers of AF. Regulation of Erg by TGF-ß was confirmed in these cells. In addition, TGF-ß soaked Affi-gel beads implanted into the axial skeleton of stage HH 25 chick embryos showed that TGF-ß could induce expression of Erg mRNA in vivo. Next, an adenovirus to over-express Erg in primary sclerotome micromass cultures was generated. Over-expression of Erg led to a change in cell morphology and inhibition of differentiation into hyaline cartilage as seen by reduced Alcian blue staining and decreased Sox9 and c-Maf expression. Erg was not sufficient to induce expression of AF markers and expression of Sca1, a marker of pluripotent progenitor cells, was up-regulated in Erg expressing cells. When cells that ectopically expressed Erg were treated with TGF-ß, enhanced expression of specific differentiation markers was observed suggesting Erg can cooperate with TGF-ß to regulate differentiation of the sclerotome. Furthermore, we showed using co-immunopreciptiation that Erg and Smad3 bind to each other suggesting a mechanism for their functional interaction.
