Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia–mutated kinase–mediated DNA damage response

Highlights

• Radiation therapy plus palbociclib exhibits synergy in liver cancer cells.

• Palbociclib treatment impairs radiation-induced double-strand break repair.

• Palbociclib blocks radiation-induced ataxia telangiectasia–mutated (ATM) kinase–dependent phosphorylation.

• Protein phosphatase 5 is involved in palbociclib-mediated ATM kinase inactivation upon DNA damage.

Abstract

Aim

Palbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) and addressed the molecular mechanism behind the combination therapy.

Methods

Immunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formation and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the in vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model.

Results

Palbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia–mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both in vitro and in vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT.

Conclusion

Our findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

Introduction

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) are the most frequently occurring types of primary liver cancer. HCC accounts for approximately 90% of primary liver cancer cases, and CCA is the second most common type of liver cancer [1], [2]. Liver cancer is currently the second leading cause of cancer-related death globally and is a neoplasm with increasing incidence (about 3% per year, from 2005 to 2014) and mortality (about 2.5% per year, from 2006 to 2015) [3]. Although some progress has been made in treating this neoplasm, the 5-year survival rate for patients with liver cancer is 18% [4], which is very low than that of other cancer types. Therefore, treatment of liver cancer remains a challenge.

Liver resection and transplantation are the main treatment options for patients in the early stages of liver cancer [5]. Patients with sufficient healthy liver tissues can sometimes be successfully treated by such surgical strategies. However, for patients at more developed stages, limited therapeutic options are available. Transarterial chemoembolization is widely used in intermediate HCC patients who are not eligible for surgery [6]. Until recently, the multi-kinase inhibitor, sorafenib (Nexavar) was the first and only approved targeted drug for patients with advanced HCC; the drug received US Food and Drug Administration (FDA) approval in 2007. However, in 2017, the FDA approved regorafenib (Stivarga) as a second-line therapy for patients who stopped responding to sorafenib [7]. Even though the aforementioned regimens have shown some survival benefits for HCC patients at the advanced stage, these drugs just extend the average survival of patients for about 3 months. In comparison with HCC, CCA is a rare malignancy; however, it is often discovered at an advanced stage. There is no approved targeted drug for CCA to date, and cytotoxic chemotherapy remains the major choice of treatment for CCA [8]. To improve the current management of liver cancers, it is imperative to develop new therapeutic strategies and combinations for use in the adjuvant setting for advanced-stage disease.

Palbociclib (PD-0332991; IBRANCE) is a small-molecule cyclin-dependent kinase 4/6 (CDK4/6) inhibitor that has shown great efficacy in treating oestrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer patients [9]. In addition to breast cancer, the potential application of palbociclib as a single therapy or in combination with other therapeutic strategies is under active investigation for numerous malignancies in both clinical and pre-clinical settings [10]. For the treatment of liver cancer, we and others have reported potent anti-HCC activity of palbociclib alone or in combination with sorafenib in preclinical models of HCC [11], [12]. Notably, palbociclib has been tested in an HCC clinical trial (NCT01356628). As a single agent, palbociclib is well tolerated in patients with advanced HCC but showed only modest activity [13], indicating that combinatorial administration of palbociclib with other therapies may be required to improve clinical efficacy for treating liver cancer.

Radiation therapy (RT) is the application of high-energy radiation to kill cancer cells and shrink tumours. RT kills cells by damaging DNA directly or indirectly via creating free radicals within the cells which in turn damage the DNA [14]. DNA double-strand breaks (DSBs) induced by RT can lead to cell death if the damage remains unrepaired [15]. Thus, interfering with the DSB repair pathway has been an attractive strategy to sensitise cancer cells to radiation or DNA-damaging chemoagents [16]. The major pathway for the repair of RT-induced DSB includes homologous recombination (HR) and non-homologous end joining (NHEJ), in which the phosphatidylinositol 3-kinase (PI3-K)–like kinases, ataxia telangiectasia mutated (ATM) kinase and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), respectively, play central roles [17]. A number of preclinical studies have demonstrated that inhibition of DSB repair proteins such as ATM kinase or DNA-PKcs could hypersensitise cells to ionising radiation and DNA DSB-inducing agents [18], [19]. There are many DSB repair inhibitors in development; however, only few have reached clinical phase trials to date [20].

Recent studies have identified palbociclib as a radiosensitizer for treating human atypical teratoid rhabdoid tumour and glioblastoma and demonstrate that palbociclib could augment the effect of RT via inhibiting the DNA damage repair process [21]. On the other hand, we previously reported that palbociclib possesses kinase-independent anti-HCC activity by inhibiting an oncogenic protein phosphatase 5 (PP5) [11]. As PP5 is known to participate in DNA damage response (DDR) by regulating key upstream DDR kinases including ATM kinase, ATR (ATM and Rad3 related) and DNA-PKcs [22], [23], [24], [25], [26], we hypothesised that palbociclib might attenuate ionising irradiation (IR)–induced DSB repair machinery via inhibiting PP5, thus sensitising cancer cells to RT. To test our hypothesis, in the present study, we addressed whether and how palbociclib could potentiate the efficacy of RT in treating liver cancer. Our results showed that palbociclib can delay the kinetics of DNA repair and enhance the radiosensitivity of HCC and CCA cells. Mechanistically, palbociclib can suppress activation of ATM kinase after IR exposure by inhibiting PP5. The inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism of the combination treatment both in vitro and in vivo. Our results show that palbociclib acts as a novel DSB repair inhibitor via targeting the PP5-ATM axis and reveal the potential utility of palbociclib as a radiosensitizer for liver cancer in the clinic.