Epigenetic reprogramming of cell cycle genes by ACK1 promotes breast cancer resistance to CDK4/6 inhibitor

Abstract

Hormone receptor-positive, HER2-negative advanced breast cancers exhibit high sensitivity to CDK4/6 inhibitors such as palbociclib. However, most patients inevitably develop resistance, thus identification of new actionable therapeutic targets to overcome the recurrent disease is an urgent need. Immunohistochemical studies of tissue microarray revealed increased activation of non-receptor tyrosine kinase, ACK1 (also known as TNK2) in most of the breast cancer subtypes, independent of their hormone receptor status. Chromatin immunoprecipitation studies demonstrated that the nuclear target of activated ACK1, pY88-H4 epigenetic marks, were deposited at cell cycle genes, CCNB1, CCNB2 and CDC20, which in turn initiated their efficient transcription. Pharmacological inhibition of ACK1 using its inhibitor, (R)-9b dampened CCNB1, CCNB2 and CDC20 expression, caused G2/M arrest, culminating in regression of palbociclib-resistant breast tumor growth. Further, (R)-9b suppressed expression of CXCR4 receptor, which resulted in significant impairment of metastasis of breast cancer cells to lung. Overall, our pre-clinical data identifies activated ACK1 as an oncogene that epigenetically controls the cell cycle genes governing the G2/M transition in breast cancer cells. ACK1 inhibitor, (R)-9b could be a novel therapeutic option for the breast cancer patients that have developed resistance to CDK4/6 inhibitors.

Fig. 1. ACK1 is activated in multiple types of breast cancers.

A Immunohistochemical analysis of human breast cancer tissue samples and normal parenchyma was performed using pY284-ACK1 and total ACK1 antibodies. Tumor status was confirmed by H&E staining (n = 500 all groups; a representative image is shown). Relative pairwise abundance of total ACK1 and pY284-ACK1 displayed by TMA staining for (B) all breast cancer samples, (C) ER⁺, (D) ER⁺/PR⁺, (E) HER2⁺, and (F) TNBC samples is shown.

Fig. 2. ACK1 regulates breast cancer transcriptome, affecting genes implicated in cell cycle progression.

A Chemical structure of (R)-9b. B Breast cancer cell lines were treated with either vehicle or (R)-9b overnight and cell lysates were subjected to immunoprecipitation using ACK1 antibody, followed by immunoblotting with pY284-ACK1 antibody. Lysates were also subjected to immunoblotting with total ACK1 and Actin antibodies, shown in the lower panels. Densitometric measurement of protein abundance relative to control is displayed below each blot. C Breast cancer cell lines were treated with varying concentrations of (R)-9b for 96 h and the cell proliferation was measured using Trypan Blue exclusion method. D–H RNA from breast cancer cells treated with vehicle or varying concentrations of (R)-9b overnight, and were subjected to qRT-PCR using CCNB1, CCNB2 and CDC20 primers. Actin or 18S rRNA was used as housekeeping control. For (B), representative images of three independent experiments are shown. For (C), a four parameter, variable slope, non-linear regression curve was computed, representing three independent experiments. For (D–H), data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test.

Fig. 3. ACK1 inhibitor suppresses expression of cell cycle genes.

A Cell lysates from vehicle or (R)-9b treated cells were immunoblotted using CCNB1, CCNB2 and CDC20 antibodies. B HCC-1395 and MDA-MB-231 cells were transfected with ACK1 and scrambled siRNAs, followed by qPCR using ACK1 primers, with 18S rRNA as internal control. C siRNA transfected cells were subjected to western blotting using ACK1 antibody, with Actin as loading control. RNA from silenced cells, (D) HCC-1395 and (E) MDA-MB-231 was subjected to qPCR using CCNB1, CCNB2 and CDC20 primers. 18S rRNA was used as housekeeping control. For (A) and (C), representative images of three independent experiments are shown. For (B, D, E), data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test.

Fig. 4. ACK1 epigenetically modifies the chromatin landscape by depositing pY88-H4 activating marks, regulating cell cycle genes.

A Breast cancer cell lines were treated with (R)-9b overnight and the cell lysates were subjected to immunoprecipitation using pY88-H4 antibody, followed by immunoblotting with H4 antibody. Lysates were also subjected to immunoblotting with total H4 and Actin antibodies, shown in the lower panels. Densitometric measurement of protein abundance relative to control is displayed below each blot. B Venn diagrams summarizing the overlap between sites bound by pY88-H4 in vehicle treated and (R)-9b treated MDA-MB-453 cells. C ChIP-Seq using pY88-H4 antibody in MDA-MB-453 revealed peaks in CCNB1, CCNB2 and CDC20 genes. Breast cancer cells were subjected to ChIP using pY88-H4 antibody followed by qPCR for (D) CCNB1, (E) CCNB2, and (F) CDC20 genes. For (A), representative images of three independent experiments are shown. For (D–F), data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test.

Fig. 5. ACK1 inhibition leads to G2/M cell cycle arrest in breast cancer cells.

A–F Breast cancer cells were treated with either vehicle or (R)-9b for 48 h; cells were harvested and processed for cell cycle analysis using propidium iodide staining, followed by flow cytometry. G BT549 and H MDA-MB-468 cells were treated with varying drug concentrations of palbociclib or (R)-9b for 96 h and the cell viability assessed using trypan blue exclusion method. I BT549, and J MDA-MB-468 cells were treated with 1 µM palbociclib or (R)-9b for 48 h and subjected to cell cycle analysis using propidium iodide staining, followed by flow-cytometry. Palbociclib-resistant MDA-MB-231 (K) and HCC-1395 (L) were generated and treated with varying drug concentrations of palbociclib or (R)-9b for 96 h and the cell viability assessed using trypan blue exclusion method.

Fig. 6. (R)-9b inhibits breast cancer xenograft tumor growth in vivo.

A 2 × 10⁶ Cal-148 cells were injected in the 4th inguinal mammary fat pad of female SCID mice. Once the tumors were palpable, the mice were treated subcutaneously with either vehicle (Captisol; n = 8) or (R)-9b at 24 mg/Kg (n = 8) five times a week, for 4 weeks. The tumor volumes were measured twice a week. B, C The tumors were harvested and photographed, and the weights were recorded. D RNA was prepared from the tumors, followed by qRT-PCR to determine the levels of CCNB1, CCNB2 and CDC20 mRNA (n = 3 each). E and F 2 × 10⁶ MCF7 cells were injected in the 4th inguinal mammary fat pad of female SCID mice with continuous supplementation of estrogen. Once the tumors were palpable, the mice were treated with vehicle (Captisol; n = 7), (R)-9b at 24 mg/Kg subcutaneously (n = 8), or orally with (R)-9b at 36 mg/Kg (n = 8), five times a week, for 4 weeks. The tumors were harvested, their weights were recorded and photographed. G Tumor lysates were subjected to immunoblotting using pACK1, CCNB1, CCNB2, CDC20 and Actin antibodies (n = 3 each). H RNA was prepared from the tumors, followed by qRT-PCR to determine the levels of CCNB1, CCNB2 and CDC20 mRNA (n = 3 each). Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. For (C) and (D), unpaired two-tailed Student’s t test, and for (E) and (H), one way ANOVA was performed.

Fig. 7. ACK1 inhibitor overcomes palbociclib-resistant breast cancer growth and metastasis.

A 3 × 10⁶ MDA-MB-468 cells were injected in the 4th inguinal mammary fat pad of female SCID mice. Once the tumors were palpable, the mice were treated with either vehicle (Captisol; n = 7), Palbociclib at 80 mg/kg (n = 6), or (R)-9b at 36 mg/Kg (n = 6) orally. In addition, mice were injected with (R)-9b at 24 mg/Kg (n = 7) subcutaneously. Mice were treated five times a week, for 22 days. B, C The tumors were harvested and photographed, and the weights were recorded. D Vehicle or (R)-9b treated breast cancer cells were subjected to ChIP using pY88-H4 antibody, followed by qPCR for CXCR4 gene. E CXCR4 mRNA transcript levels in vehicle or (R)-9b treated MDA-MB-231 cells. 18S rRNA was used as housekeeping gene. F MDA-MB-231 cells were transfected with ACK1 or scrambled siRNA, followed by qRT-PCR to determine CXCR4 mRNA transcript levels. G Representative lung metastatic deposits of MDA-MB-231 cells injected via the tail veins in female SCID mice, treated with either vehicle or 24 mg/Kg (R)-9b orally. H Graphical quantitation of lung metastatic deposit. I and J 5 × 10⁵ luciferase expressing 4T1 cells were injected into 4th inguinal mammary fat pad of female BALB/c mice. Once the tumors were palpable, mice were treated with either vehicle or (R)-9b at 24 mg/Kg (n = 3, each) subcutaneously for 2 weeks, five times a week. The metastases were assessed using IVIS. Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. For (A, C–F, I), p values were calculated using unpaired two-tailed Student’s t test.