Synergistic anti-cancer activity of CDK4/6 inhibitor palbociclib and dual mTOR kinase inhibitor MLN0128 in pRb-expressing ER-negative breast cancer

Abstract

Purpose: Palbociclib is an approved cyclin-dependent kinase (CDK) 4/6 inhibitor for treatment of patients with ER-positive and HER2-negative breast cancers. While Retinoblastoma protein (pRb), a major substrate of CDK4/6, is a potential target in triple negative breast cancer (TNBC), the usefulness of CDK4/6 inhibitors in this cancer has not been established. This preclinical study investigated the combination effects of palbociclib and the dual mammalian target of rapamycin (mTOR) kinase inhibitor MLN0128 in estrogen receptor (ER)-negative breast cancer in vitro and in vivo.

Methods: The combined effects of two drugs on three TNBC cell lines (MB231, MB468, and CAL148) and an ER-negative and HER2-positive cell line (MB453) were investigated by MTT assay and colony formation analysis. Cell cycle measurements were examined as well as changes in expression of molecules related to G1/S transition and the mTOR pathway. Importantly, a pRb-expressing TNBC patient-derived xenograft (PDX) model was used to assess the effects of the combination in vivo.

Results: A combination of palbociclib and MLN0128 synergistically inhibited the proliferation of pRb-expressing cell lines and induced G1 cell cycle arrest. Western blot analysis revealed that CDK4/6-pRb and mTOR pathways were inhibited by these treatments. In pRb-expressing TNBC PDX, the combination treatment drastically suppressed tumor growth compared to either the control or single drug treatments. In addition, the combination treatment significantly reduced the number of Ki67-positive cells.

Conclusions: We revealed that palbociclib and MLN0128 had synergistic anti-cancer activity in both pRb + ER-negative cell lines and a TNBC PDX model. Our results indicate that such combination therapy is worthy of further investigation in a clinical setting.

Keywords: CDK 4/6 inhibitor; MLN0128; Palbociclib; Retinoblastoma protein (pRb); Triple negative breast cancer (TNBC); mTOR inhibitor.

Fig. 1:. Palbociclib suppressed proliferation of pRb-expressing TNBC cells.

(A) Cell lysates were evaluated for phospho-pRb and pRb expression by Western blot. β-actin is shown as loading control. (B) MB231 and MB453 cells, and (C) MB468 and CAL148 were treated with palbociclib at the indicated concentrations and cell proliferation was assessed by MTT assay after 24, 72, and 120 h incubation. Results of MTT assay are expressed as the ratio of control at the time, and are mean values ± SD at each palbocilcib concentration, from triplicate measurements for each treatment.

Fig. 2:. Combination of palbocilclib and MLN0128 synergistically inhibited proliferation of MB231 and MB453.

(A and C) MB231, MB453, MB468, and CAL148 cells were treated with palbociclib and/or MLN0128 for 120 h. The X axis shows palbociclib concentration. (B) Combination index (CI) was calculated in MB231 and in MB453. CI > 1 means antagonistic effect, = 1 means additive effect, and < 1 means synergistic effect. (D) MB231 and MB453 cells were treated with palbociclib and/or everolimus at the indicated concentrations. Results of MTT assay are expressed as the ratio of control, and are mean ± SD at each treatment concentration, from triplicate measurements for each treatment.

Fig. 3:. The combination treatment suppressed colony formation ability in MB231 and MB453 cells.

(A) MB231 cells were plated into 6-well plates in 2 ml complete media at 1000 cells/well. Cells were treated with or without palbociclib at 250 nM and/or MLN0128 at 31.25 μM for 7 days. MB453 cells were plated at 2000 cells/well and treated with or without palbociclib at 62.5 nM and/or MLN0128 at 7.8125 nM for 21 days. (B) Colony numbers from the four treatment groups were counted. Data are mean values ± SD, from duplicate measurements for each treatment. Statistic analyses were subjected unpaired t test compared to the combination treatment. *p < 0.05, **p < 0.01.

Fig. 4:. The combination induced G1 arrest and reduced G1/S trantision related molecules and mTOR pathway.

MB231 cells were treated for 48 h with or without palbociclib at 250 nM and/or MLN0128 at 31.25 μM. MB453 cells were treated for 48 h with or without palbociclib at 62.5 nM and/or MLN0128 at 7.8125 nM. (A) Flow cytometric analyses were performed in MB231 and MB453. Data are expressed as cell population (%) and mean values ± SD, from duplicate measurements for each treatment. Statistic analyses of G1 cell population were subjected unpaired t test compared to control or combination treatment. *p < 0.05, **p < 0.01. (B and C) Lysates were prepared from cells from each traetment and evaluated through Western blot analysis. Arrows indicate the target bands. β-actin is shown as the loading control. (D) Cyclin E1 mRNA levels in MB231 and MB453 were mesaured by realtime PCR. ACTB was used as a reference. Data are expressed as the ratio of control and mean values ± SD, from triplicate measurements for each treatment. **p < 0.01.

Fig. 5:. Palbociclib in combination with MLN0128 showed a cooperative anti-tumor effect in pRb-expressing TNBC PDX tumors.

(A) Expressions of phospho-pRb and pRb were examined by Western blot in 3 different untreated tumors of COH_GS6. (B) Mice bearing COH_GS6 tumor received control vehicle, MLN0128, and/or palbociclib. All vehicle and drugs were administered 6 days each week. Data of tumor growth rate are expressed as the percentage of each tumor size on day 0, and are mean ± SEM. Tumor volume was monitored once a week. (n = 7) (C) Tumors were collected and their weights were measured on day 39. Black bars show mean values and the error bars represent SEM. (D) Pictures of tumors were taken when tumors were collected. The scale (cm) has been placed below the tumors. (E) Mice body weight was monitored at the time of treatment. Each color shows the body weight of each group of mice. Data are mean values ± SEM. (F) Tumor lysates were prepared. Tumors were collected 6 h after oral gavage of drugs at the last day. Western blot was performed using indicated antibodies. β-actin is shown as the loading control. (G) Representative image of IHC of Ki67 are shown for each treatment condition. A scale bar of 200 μm is shown below the images. (H) Data are expressed as the percentage of Ki67 positive cells in harvested tumors receiving each treatment [palbociclib group (n = 3), and other groups (n = 4)]. The boxes show the first quartile and third quartile of data and the lines in the box show median values. The bars at the end of box show 1.5 IQR. Statistic analyses were subjected to one-way ANOVA with Tukey’s multiple comparison post-hoc test for data of (B), (C), and (H), and unpaired t-test for data of (E). *p < 0.05, **p < 0.01, ***p = 0.001.