GPR81 nuclear transportation is critical for cancer growth and progression in lung and other solid cancers

Figure 4 Inhibition of GPR81 nuclear translocation reduces cancer cell self-renewal and invasion. Lung cancer cells were transduced with empty vector, GPR81 wild-type or NLS mutant and cultured under vehicle or 5 mmol/L lactate conditions. A: Lung cancer cell self-renewal was assessed in the colony forming assay; B: Cancer stemness marker CD44 and proliferation marker Ki67 were quantified with reverse transcriptase-PCR (RT-PCR) and western blot analysis; C: Lung cancer cells invasion was assessed; D: Cell motion marker MMP2 was quantified in RT-PCR and western blot analyses using cell lines H549, H661, H848 and H448. Data are shown as mean ± SE. 3 technical replicates were performed for all experiments. GPR81 translocation affects lung cancer progression in vivo. NSG mice implanted with lung cancer cells (A549, H848 and H661) stably transduced with either empty vector, GPR81 NLS mutant, or wild-type GPR81 via IV (3 × 10⁵ cells/50µl); 6 mice/group; E-P: Serial 4 µm sections of right lung tissue from lung cancer tumors transduced with GPR81NLS mutant or wild-type GPR81. Representative H&E (E-G; scale bar: 500 µm) and immunohistochemistry (IHC) stains assessing GPR81, Ki67 and MMP2 positivity. IHC identifies GPR81, Ki67 and MMP2 and determines the distribution of GPR81 expression cells (H-J), Ki67 expressing cells (K-M) and MMP2 expressing cells (N-P). IHC for GPR81 and Ki67 was conducted to assess the distribution of human cells expressing GPR81, Ki67 and MMP2 protein expressing cells from mice from tumors of lung cancer cells transduced with empty vector, GPR81 wild-type (GPR81wt) or GPR81 NLS mutant (GPR81NLSmu). Inner frame shows an enlarged image from the indicated area. Scale bar = 200 μmol/L; Q-S: GPR81, Ki67 and positive cells in lung cancer cell explanted mice lung tissue sections; T: Cell quantification was conducted with quantitative PCR.