26 Background: CD98, a transmembrane protein, has a heteromeric structure, consisting of a heavy subunit (CD98hc) and a light subunit, extracellular linked together via disulfid bounds. A genetic knockout of CD98hc is embryonic lethal and overexpression of CD98hc in somatic cells led to malignant transformation. CD98hc is highly expressed in low differentiated papillary, clear cell and chromophobe RCC, but not in benign tumors. Notably, CD98hc expression directly correlated with grade of differentiation. Methods: To evaluate a potential functional role of CD98hc in renal cancer cell metastatic behavior, we generated a stable low CD98hc clear cell RCC cell line (Caki2) via lentiviral shRNA infection and compared tumor cell behavior with a high expressing mock transfected control. Results: We found that tumor cell behavior such as proliferation (52 ± 3% less 3[H] thymidin – incorporation in low CD98hc/Caki2 cells), cell survival upon anoikis (46% late and 45% early apoptosis in low CD98hc/Caki2 cells compared to high/CD98hc/Caki2 cells with 18% late apoptosis and 64% early apoptosis) and invasion/transmigration (520 ± 67 cells / field after 24h in low CD98hc/ Caki2 cells and 1257 ± 346 cells / field in high CD98hc/Caki2 cells analyzed in a modified boyden chamber) were considerably impaired whenever CD98hc expression was downregulated. To examine the mechanism by which CD98hc affected metastatic tumor cell behavior, we introduced two mutations in a reconstitution (silent mutation). A truncation mutant interfered with integrin interaction and a two point mutations (Cys109Ser and Cys330Ser) mutant affected amino acid transporter activity. Whenever integrin/CD98hc interaction was impaired tumor cell behavior including cell proliferation, cell survival, invasio/transmigration, and cell spreading as well as signal transduction pathways including FAK, c-src, MEK/ERK pathways were significantly compromised resembling the low CD98hc phenotype. Conclusions: For these data we conclude that metastatic tumor cell behavior such as cell survival, invasion and proliferation are dependent on CD98hc expression in renal cancer cells.
Hepatocyte-tumor cell interaction in vitro. I. Conditions for rosette formation and inhibition by anti-H-2 antibody.
