Figure 5. Synergism between palbociclib and autophagy inhibition in other solid tumours.

(a) Western blot for Rb and Cyclin E in TNBC cell lines. (b) IC₅₀ values from drug response experiments in TNBC cell lines treated with palbociclib for 6 days. (c) Quantification of GFP-LC3 puncta and representative images in MDA-MB-231 cells treated with 1 μM palbociclib and/or 25 μM CQ for 48 h. TNBC cell lines were treated with combination of 1 μM palbociclib and 15 μM HCQ for 6 days and subjected to (d) Clonogenic and (e) SA-ß galactosidase assays. (f) Percentage change in volume (normalized to Day 0) of PDX tumours upon treatment with Vehicle, 25 mg kg⁻¹ palbociclib, 60 mg kg⁻¹ HCQ or combination of palbociclib (25 mg kg⁻¹) and HCQ daily for 21 days. Data represented as mean±s.e.m. n=4 for each group. (g,h) Kaplan–Meier survival curve with death and tumours exceeding 1,000 mm³ as end point (g) and weight (h) of PDX tumours treated as in (f). n=4 for each group. (i) Western blot of Rb and Cyclin E in ovarian, pancreatic (PDAC), lung, colon and prostate cancer cell lines. (j,k) IC₅₀ values from drug response experiments (j) and Clonogenic assay (k) in the mentioned cancer cell lines treated with palbociclib for 6 days and recovery for 6 days. (l,m) Cell counting (l) and clonogenic assay (m) in cancer cell lines treated with 1 μM palbociclib and 15 μM HCQ for 6 days and recovery for 4 or 6 days respectively. P value calculated in comparison with 1 μM palbociclib. (n) Correlation between palbociclib IC₅₀ values (from dose–response studies in all cancers) and levels of Rb and cyclin E proteins, with and without inhibition of autophagy (Beclin-1/Atg5 knockdown or HCQ treatment). (o) Schematic depicting the mechanism by which palbociclib inhibits growth of Rb+/LMWE− breast cancer cells by regulating ROS, autophagy and senescence. All data represent mean±s.d. from three independent experiments; NS: P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.