Meredith Lomeli

Lung cancer is the leading cause of cancer-related deaths worldwide. The average 5-year survival rate is 15% and as low as 8% in patients with advanced-stage tumors. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancer diagnoses. Metastasis has been shown to be the primary reason for NSCLC deaths, which is assisted by oncogenic drivers and cooperation with the tumor microenvironment. However, the mechanisms behind NSCLC metastasis are not well understood. CXCL10 is a chemokine that normally binds to CXCR3, a receptor on immune cells such as T cells, to induce chemotaxis during inflammation. In some types of cancer, including breast cancer, high CXCL10 expression promotes metastasis and is thus associated with poor patient outcome. Additionally, it was found that CXCL10 expression is negatively regulated by the tumor suppressor Inhibitor of Growth 4 (ING4) in breast cancer, suggesting that CXCL10 may drive tumor progression in the absence of ING4. We demonstrated that CXCL10 induced migration, a prerequisite for metastasis, of ING4-deleted breast cancer cells. However, it is not known if CXCL10 elicits migration similarly in other cancer types. This study addresses CXCL10-induced migration in the context of ING4-deficient lung cancer. To test CXCL10 signaling in lung cancer, we genetically engineered the NSCLC cell line NCI-H23 using the CRISPR/Cas-9 system to delete the ING4 gene and validated these cell lines using western blot. To evaluate whether the absence of ING4affected cell proliferation, we conducted a colorimetric Sulforhodamine B assay. We assessed the migratory capacity of the ING4-wild type or ING4-deleted H23 cells in the presence or absence of CXCL10 using Boyden chamber migration assays. To determine whether CXCR3 was required for CXCL10-induced migration, we performed migration assays using the CXCR3 inhibitor AMG-487. The western blot results showed that the amount of ING4 protein was reduced by 75% in the ING4-deleted cells (v2h1) compared to the ING4-wild type cells (v2), indicating that the CRISPR-mediated deletion of ING4 was effective. The presence of CXCL10 did not affect ING4 expression. Further, the protein levels of CXCR3 and EGFR were comparable in both cell lines, demonstrating that neither ING4 nor CXCL10 affected their expression. The proliferation assay results showed that CXCL10 did not affect the growth rates of ING4-wild type orING4-deleted cells. The Boyden chamber migration assay indicated that CXCL10 increased cell migration of ING4-deleted cells, but not of ING4 wild type cells, suggesting that ING4 inhibited CXCL10 signaling. Inhibition of CXCR3 using AMG-487 resulted in no increase in migration of ING4-deleted cells, demonstrating that CXCR3 was required for CXCL10-induced migration. Collectively, these results indicate that CXCL10 induces migration in ING4-deleted NSCLC cells. They further suggest ING4/CXCL10 signaling as a potential therapeutic target for aggressive lung cancer.