Fig 2: Cyclin D1/CDK4 expression is correlated to the presence of KRAS mutation in lung cancer tumours. A. pRb, CDK4 and cyclin D1 immunoperoxidase nuclear staining in lung adenocarcinoma (ADC, upper panels) and in squamous cell carcinoma (SCC, lower panels). CyclinD1 (panel 1, score of 70%; panel 2, score of 40%), CDK4 (panel 3, score of 50%; panel 4, score of 50%), and pRb (panel 5, score of 10%; panel 6, score of 20%) expression. Bars, 100 µm (ADC) and 50 µm (SCC). B. Distribution of cyclin D1, CDK4, and pRb staining (percentage of positive cells, Y-axis) according to the histological subtype of tumours. Statistical analysis was carried out using Mann-Whitney's U-test. C. Cyclin D1 expression according to KRAS mutation status of tumours. Statistical analysis was performed using Fisher's exact test. Missing data were excluded from the analysis.

Fig 3: P2shortA inhibits orthotopic H358 lung tumour growth when co-administered with Abemaciclib. A. Schematic representation of H358 orthotopic tumours growth and of the treatments plan. The mice were inoculated with H358-rvLuc cells and randomized after 5 weeks into 3 groups of 10 mice and one control group of 14 mice. Vehicle or abemaciclib 10 mg/kg were administered per os every days for 3 weeks. 0.45 mg/kg P2shortA was administered intrapulmonary 2 times a week for 3 weeks. Thoracic bioluminescence imaging was performed once a week to follow tumour growth. B. Overtime thoracic bioluminescence images of H358-Luc tumours. One representative mouse (dorsal view) per group is shown. C. P2shortA and Abemaciclib combination inhibited the growth of orthotopic H358 tumours. The results are expressed as the mean ± SEM (control group, n = 14; treated groups, n = 10). Friedman test with Dunn's multiple comparisons posthoc tests (p = 0.0026); *, treatment combination group compared to control group; **, treatment combination group compared to P2shortA group. D. Thoracic bioluminescence level in each mouse at day 56 showed reduced level in P2shortA and Abemaciclib combination group. Bars, medians with interquartiles. Kruskall-Wallis test with Dunn's multiple comparisons posthoc tests; *, treatment combination group compared to control group. E. Histological (HE) and immunohistochemistry for Ki67 and phosphorylated-Rb (pRb) on lung tumour sections. Scale bars, 100 µm (HE) and 50 µm (Ki67 and pRb). F. Ki67-positive cells were quantified on different fields (2-7 fields) in three mice per group, and reported as percentage of Ki67 positive cancer cells. Bars, medians with interquartiles. Kruskall-Wallis test with Dunn's multiple comparisons posthoc tests (p = 0.0042); **, treatment combination group compared to control group. G. pRb-positive cells were quantified on different fields (2-29) in three or four mice per group, and reported as percentage of pRb positive cancer cells. Bars, medians with interquartiles. Kruskall-Wallis test with Dunn's multiple comparisons posthoc tests (p < 0.0001); ***, treatment combination group compared to P2shortA group; ****, treatment combination group compared to control or Abemaciclib groups.

Fig 4: Downregulation of CCND1b could demote cell proliferation and decelerate the cell cycle progression in BC cells. (A, B) MCF‐7/ADM cells were transfected with siRNA targeting CCND1b, and the expression of CCND1b was analysed by RT–PCR and Western blotting. (C) MCF‐7/ADM cells of control and siCCND1b underwent treatment with ADM for 48 h and analysed cell proliferation by Cell Counting kit‐8 assay. (D) Half maximal inhibitory concentration values of MCF‐7/ADM cells of control and siCCND1b for ADM are presented. (E) Cell cycle progression was investigated by propidium iodide staining and flow cytometry in MCF‐7/ADM cells of control and siCCND1b. The percentages of cells at the different phases were calculated and plotted. (F) The expression of pRB, E2F1 and CDK4 proteins after knockdown of CCND1b. The experiment is repeated and calculated in triplicate (N = 3). The data are expressed as the mean ± SD. ***p < 0.001; **p < 0.01; *p < 0.05.

Fig 5: Upregulation of CCND1b/a ratio is associated with chemoresistance via cyclin D/CDK4‐pRB pathway in BC cells. (A) mRNA levels of CCND1a and CCND1b in MCF‐7 and MCF‐7/ADM cells were analysed by semi‐quantitative RT–PCR. (B) The protein expression levels of CCND1a and CCND1b in MCF‐7 and MCF‐7/ADM cells were analysed by Western blotting. (C) Quantitative analysis of the mRNA expression of CCND1a, CCND1b and CCND1b/a ratio in MCF‐7 and MCF‐7/ADM cells. (D) Quantitative analysis of the protein expression of CCND1a, CCND1b and CCND1b/a ratio in MCF‐7 and MCF‐7/ADM cells. (E) The correlation of CCND1 with some BC‐associated drug resistance genes was analysed by the Assistant for Clinical Information online platform. (F) Model of CDK4 binding to the CCND1a and CCND1b. (G) The protein expression levels of pRB, CDK4 and E2F1 in MCF‐7 and MCF‐7/ADM cells. (H) Quantitative analysis of the protein expression of pRB, CDK4 and E2F1 in MCF‐7 and MCF‐7/ADM cells. (I) Working model of the upregulation expression of CCND1b/a ratio involved in BC chemoresistance via cyclin D/CDK4‐pRB pathway. The experiment is repeated and calculated in triplicate (N = 3). The data are expressed as the mean ± SD. ***p < 0.001; **p < 0.01; *p < 0.05.