Carlos Rossa, DDS, PhD
Role of SOCS1 and SOCS3 in Attenuating Signaling Leading to Oral Squamous Cell Carcinoma Progression (Start date 8/1/08)
High constitutive or induced levels of cytokine expression are fundamental for oncogenesis and metastasis because they affect critical processes such as proliferation, survival, angiogenesis and invasiveness. This increased cytokine expression is associated with sustained activation of signal transduction pathways, which can be the result of a defective endogenous regulatory mechanisms. Suppressors of cytokine signaling (SOCS) is a family of structurally-related proteins that can modulate JAK-STAT, MAPK and NF-kB signaling pathways and whose expression has been shown to be decreased in various types of cancer, including oral squamous cell carcinoma (OSCC). The hypothesis is that the endogenous negative regulatory mechanism of cytokine signal transduction mediated by the SOCS proteins has a crucial role on OSCC tumor progression and invasion.
Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common cancer worldwide and presents a very poor prognosis, with a survival rate reported to be less than 50%. Constitutive or increased levels of induced cytokine expression by tumor cells are considered fundamental both to oncogenesis and aggressiveness of cancer because it affects critical cell biology processes, including proliferation and survival. Accumulating evidence indicates that oral squamous cell carcinomas are characterized by increased levels of cytokine gene expression, which is the result of sustained activation of signal transduction pathways. The JAK-STAT pathway is the canonical signaling pathway utilized by cytokine receptors, and the relevant role of STAT activation has been widely demonstrated in various types of cancer, including oral squamous cell carcinoma (OSCC). However, other signaling pathways, such as MAPK and NFκB, are also constitutively activated in OSCC and have been shown to play important roles in tumor progression. These other signaling pathways are required for inflammatory cytokine gene expression, which is a process usually strictly regulated by various mechanisms including some endogenous regulators of cytokine signal transduction. Suppressor of activated cytokine signaling (SOCS) is a family of structurallyrelated proteins that has been shown to modulate both JAK-STAT signaling and/or STAT activity and, significantly, also MAPK and NF-κB signaling. Interestingly, epigenetic modification of SOCS genes resulting in decreased expression of SOCS proteins has been observed in various types of cancer, including OSCC, suggesting that impaired functioning of the SOCS-mediated regulatory mechanism may account for sustained activation of signaling and increased level of cytokine expression by the tumor cells. However, there is a paucity of information in regard to the moment of decreased SOCS expression and its role on OSCC tumorigenesis and progression/invasion. Based upon this information, our hypothesis is that the endogenous negative regulatory mechanism of cytokine signal transduction mediated by the SOCS proteins has a crucial role on OSCC tumor progression and invasion. To test this hypothesis and to obtain relevant information on the role of SOCS1 and SOCS3 proteins in OSCC tumor progression, the specific aims are:
To evaluate the expression of SOCS1 and SOCS3 in various stages of OSCC tumor progression. class="detail" The loss of function of SOCS-mediated regulatory mechanism may occur at different stages of OSCC progression. In this aim we will use commercially-available tissue microarray analysis (TMA) to assess expression of SOCS1 and SOCS3 by immunohistochemistry in OSCC, and correlate the expression levels with the stage of tumor progression. This aim will provide information regarding the relative role of SOCS-mediated negative regulation of signaling in OSCC tumorigenesis and late-stage invasion and metastasis.
To determine the effects of SOCS1 and SOCS3 on OSCC tumor cells in vitro. class="detail" Constitutive activation of various signaling pathways, including JAK-STAT, NF-kB and MEK/MAPKinase is associated with increased cell survival and cytokine expression and SOCS proteins play an important role as negative regulators of these signaling pathways.
Sub-aim 2.1 We intend to use a gene therapy approach to over express SOCS1 and SOCS3 and reciprocal studies using RNAi approaches to knock down SOCS1 and SOCS3 expression to evaluate their impact on invasiveness, apoptosis and proliferation of OSCC cell lines in vitro using flow cytometry and invasion assays.
Sub-aim 2.2 Since SOCS1 and SOCS3 have been shown to affect other signaling pathways such as MEK/MAPkinase and NF-kB, which are critical for cytokine gene expression and tumor invasion/metastasis, we intend to determine the effects of SOCS1 and SOCS3 expression on the activation of these signaling pathways and also on cytokine gene expression by RT-qPCR and western blot assays. The physical interaction between SOCS1 and SOCS3 and other signaling intermediates both known and potentially novel will be determined by immunoprecipitation followed by mass spectrophotometry analysis.
To assess the role of SOCS1 and SOCS3 in OSCC tumor progression in a xenograft model in vivo. class="detail" An established orthotopic floor-of-mouth model for OSCC growth and spread will be used. This model was recently published  and optimized for a variety of human OSCC cell lines to consistently produce tumors of 2-4 mm (35-60 mm3) after approximately 2-4 weeks of injection in the floor-of-mouth region. Since low tumor cell dose and injection volumes are used, airway compromise due to tumor growth is minimized and allows long-term (8-10 weeks) study of tumor growth in vivo. The use of nude athymic mice instead of severe combined immunodeficent (SCID) mice allows for the use of human cell lines whereas still in the presence of NK and B cells, which are important sources of cytokines that play a role in tumor-stroma interaction. The model is especially suitable to correlate in vitro findings with in vivo tumor progression and evaluation of potential therapeutic strategies. After the primary tumor is readily observable by visual inspection and palpation, adenoviral particles encoding tagged SOCS1 and SOCS3 proteins will be injected into the tumor. Tumor growth will be monitored by direct measurements in vivo, whereas metastasis, expression of cytokine genes relevant to tumor invasion, and activation of signaling pathways will be studied by RT-qPCR, ELISA, western blot and histological/immunohistochemical methods. These studies will define the role of SOCS1 and SOCS3 proteins in OSCC tumor progression and provide additional information in regard to the relative role of SOCS proteins in oncogenesis and tumor progression. The proposed studies will also determine the effects of SOCS1 and SOCS3 on signal transduction pathways relevant for cytokine gene expression and identify novel binding partners of SOCS1 and SOCS3. The results can provide potential novel targets for therapeutic approaches aiming at inhibition of OSCC tumor progression and will be used for the elaboration of a new investigator-initiated R01-type proposal to investigate the mechanisms of SOCS-mediated regulation of signaling in OSCC. Internal support for this pilot proposal by the COBRE Grant mechanism is especially critical, since the PI on this proposal not only is a new investigator (never before funded by NIH) but is also changing the focus of his research interests from periodontal disease immunology to oral cancer immunology.