Abstract A27: Hypoxia-inducible factor 1-alpha suppression drives recruitment of metastasis-promoting granulocytes through the CXCR2 axis in renal cell carcinoma
Authors: Schokrpur Shiruyeh , Moughon Diana , Sung James L. , Xu Jingying , Wu Lily 
Published: May 2015

Abstract
Introduction: Neutrophil infiltration into localized clear cell renal cell carcinoma (RCC) tumors has been shown to be associated with reduced survival in the clinic, though their protumorigenic role in this context is unclear. Molecularly targeted therapies for metastatic RCC, such as sunitinib and sorafenib, eventually result in resistance and disease progression concurrent with increased granulocytic infiltration. Our studies aim to elucidate the metastasis-promoting role of granulocytes in RCC.
Interleukin 8 (IL-8) upregulation is one described mechanism whereby RCC tumors establish resistance to sunitinib. IL-8 is a CXCR2 ligand involved in the recruitment of granulocytes. Recent studies demonstrate that CXCR2 recruited polymorphonucler myeloid-derived suppressor cells (PMN-MDSCs), a population of immature granulocytes, promote metastasis through various mechanisms. Hypoxia-inducible factor 1-alpha (HIF-1α) suppression leads to increased expression of CXCR2 ligands, such as IL-8. These findings led us to hypothesize that suppression of the HIF-1α pathway, which is the target of current clinical therapies in RCC, stimulates the recruitment of metastasis-promoting granulocytes through upregulation of CXCR2 ligands in RCC.
Experimental Procedures: The RENCA cell line was utilized for our studies, due to its propensity to spontaneously metastasize when orthotopically implanted in BALB/c renal capsules. Levels of HIF-1α were knocked down using lentivirus harboring a short hairpin RNA (shRNA) to this gene. RENCA cells were also transduced to express green fluorescent protein (GFP) and Renilla luciferase, for tracking metastases. We confirmed HIF-1α knockdown by Western blot and RT-PCR and evaluated CXCR2 ligand expression by RT-PCR. An in vitro migration assay was utilized to assess increased recruitment of granulocytes. These analyses were extended in vivo in subcutaneous, orthotopic and tail vein RENCA models. Finally, we tested for increased metastases using histology and flow cytometry.
Results: Preliminary work has revealed that HIF-1α suppression by shRNA knockdown leads to a significant upregulation in gene expression of CXCR2 ligands CXCL1 and CXCL2 in the RENCA murine RCC line. This upregulation was shown to be functionally significant, as recruitment of granulocytes from peripheral blood was increased toward RENCA cells with suppressed HIF-1α as compared to control cells. This increased recruitment was validated to be CXCR2-dependent through the use of SB225002, a small molecule inhibitor of CXCR2. Tumor infiltration by granulocytes was increased in the HIF-1α knockdown RENCA tumors compared to control tumors in both subcutaneous and orthotopic models. A small orthotopic pilot study revealed increased metastatic spread of HIF-1α knockdown RENCA cells to the lungs compared to control cells, but a tail vein model of metastasis revealed a reduced tumor burden in the lungs of animals injected with HIF-1α knockdown cells compared to control cells. This indicates that increased metastasis in the HIF-1α knockdown cells might be occurring, but would have to be at the level of escape from the primary tumor.
Conclusions: Our findings suggest that HIF-1α suppression does lead to increased recruitment of granulocytes in the primary tumor in a CXCR2-dependent manner, and that the step of metastasis aided by these cells is likely at the level of primary tumor escape. Future work will focus on elucidating the pathway whereby CXCR2 ligands are upregulated by HIF-1α suppression, finding how granulocytes may be contributing to increased cell motility and aggressiveness in vitro and confirming an increased metastatic phenotype in the orthotopic model.
The rationale for the proposed research is that once it is known that granulocytes can contribute to disease progression, these cells can be targeted in conjunction with current clinical therapies to improve prognoses for patients with metastatic RCC.
Citation Format: Shiruyeh Schokrpur, Diana Moughon, James L. Sung, Jingying Xu, Lily Wu. Hypoxia-inducible factor 1-alpha suppression drives recruitment of metastasis-promoting granulocytes through the CXCR2 axis in renal cell carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A27. doi:10.1158/1538-7445.CHTME14-A27