MVP and the Arf6 pathway cooperatively promote cancer malignancy. (A and B) MDA-MB-231 or MDA-MB435s cells, pretreated with siRNAs for EPB41L5, GGT-II, Rab11b, or Irr, were incubated with the indicated doses of Gemcitabine (A) and Temsirolimus (B) for 72 h, and their viabilities were then measured as described in the Cell lines section within Materials and methods. (C and D) Breast cancer cells were incubated with Gemcitabine (C) or Temsirolimus (D), with or without Simvastatin, as indicated. After incubation for 72 h, their viabilities were measured. At each point, concentrations of Simvastatin were equal to those of Gemcitabine or Temsirolimus. Each experiment was performed at least twice. (A–D) The results represent mean ± SEM from three independent experiments measuring 3 wells per condition in each experiment. (E–G) High expression levels (top 33%) of HMGCR mRNA alone (E), together with high expression levels of mRNAs of components of the Arf6 pathway (F), or together with high expression levels of EPB41L5 mRNA (G) in primary breast tumors were examined with regard to the poor overall survival of patients using the Cancer Genome Atlas RNASeq dataset (n = 970). Others, patients without the simultaneous high expression of HMGCR and Arf6 pathway component mRNAs (F) or patients with expression level combinations of EPB41L5 and HMGCR other than EPB41L5-high and HMGCR-high (G). The survival rates of each group are shown as Kaplan-Meier curves, in which p-values represent the results of the log-rank test. *, P < 0.05; **, P < 0.01.