Palbociclib inhibited cell cycle progression in NPC cell lines
Previous studies have established p16 inactivation, cyclin D1 overexpression, and functional RB as predictors of palbociclib sensitivity in cancer cells (22,24,25,34). We first examined the levels of p16, cyclin D1, phospho-RB-Ser780 (an indicator of the functional status) and other relevant proteins in lysates of NPC cell lines and immortalized NPE cell lines grown in both 2D monolayer and 3D spheroid cultures for 3 days (Fig. 1A). Notably, p16 protein was detected in the immortalized NPE cell line (NP69) but not the NPC cell lines (C666-1, C17 and NPC43). RB (Ser780) phosphorylation was detected in all the NPE and NPC cell lines, albeit at variable levels, and was closely associated with the expression of cyclin A, an indicator of the proliferation status. Cyclin D1 and CDK4 were universally detected in all the NPE and NPC cell lines (Fig. 1A).
We next examined whether 2D-cultured NPC cell lines and non-malignant NPE cell lines would exhibit different levels of sensitivity to palbociclib. All cell lines were treated with 0.2-µM palbociclib for 24 hours, and changes in the levels of pRB, total RB and cyclin A from before and after treatment were monitored. As shown in Fig. 1B, treatment with palbociclib strongly reduced the levels of pRB in all three NPC cell lines but not in the immortalized NPE cells. Furthermore, the levels of cyclin A, a marker of cell cycle entry, were reduced in all three NPC cell lines after treatment with palbociclib, whereas this drug had no significant effect on cyclin A protein expression in the three immortalized NPE cell lines. A flow cytometric cell cycle analysis verified that a 24-hour treatment with 0.2-µM palbociclib induced significant G1 arrest in all three NPC cell lines but had no significant effect on cell cycle progression in the three immortalized NPE cell lines (Fig. 1C). The proportions of cells in G1 increased by approximately 0%, 14%, and 6% in the immortalized NPE cell lines NP69, NP361, and NP460, respectively, after palbociclib treatment, compared to dramatic increases of 81%, 55%, and 53% in the NPC lines C666-1, C17, and NPC43, respectively, after treatment. These results suggest that NPC cells are more susceptible than NPE cells to the inhibitory effects of palbociclib.
Low-dose and high-dose palbociclib exhibited cytostatic and cytotoxic effects, respectively, in NPC cell lines
We then evaluated the responses of the three NPC cell lines in response to various palbociclib concentrations over different time periods. The dose–response curves of C666-1, C17, and NPC43 are shown in Fig. 1D. The IC50 values of palbociclib in NPC43 at Days 1, 3, and 5 of treatment (50.07, 24.63, and 17.15 µM, respectively) were generally higher than those of C666-1 (35.67, 22.36, and 10.53 µM, respectively) and C17 cells (36.12, 20.23, and 12.75 µM, respectively). We further compared these IC50 values to the reported IC50 values of palbociclib in other cancer cell lines (adapted from the Genomics of Drug Sensitivity in Cancer databank, Supplementary Fig.S1) (35). A mean IC50 of 35.7 µM on Day 3 of treatment was calculated for 770 cancer cell lines. Twenty-six of these cell lines were head-and-neck cancer cell lines that had a mean IC50 of 49.2 µM on Day 3, suggesting that our tested NPC cell lines may be more sensitive to palbociclib treatment than other head and neck cancer cell lines.
We also observed that palbociclib induced cytostatic effects in NPC cells at low doses (8 nM–5 µM) and cytotoxic effects at high doses (20–40 µM) (Fig. 1E). Cleaved-PARP, an apoptosis marker, was only detected in NPC cells treated with high-dose palbociclib (20, 20, and 40 µM for C666-1, C17, and NPC43, respectively) (Fig. 1E, Supplementary Fig. S2). However, we observed potent suppression of cyclin A protein expression and RB-Ser780 phosphorylation in cells treated with much lower doses of palbociclib. We also examined the growth inhibitory effect of palbociclib in 3D cultures of C666-1 and C17 (Supplementary Fig. S3) and confirmed the suppression of cyclin A expression and RB-Ser780 phosphorylation in these NPC spheroids.
We further assessed the cell cycle distribution of NPC43 cells exposed to different concentrations of palbociclib from Days 1 to 5 (Fig. 1F). A sub-G1 peak indicative of cellular apoptosis was only observed in NPC cells treated with high-dose palbociclib (20 µM). However, G1 arrest was observed even at lower doses of palbociclib, which further supports the distinct dose-dependent effects of palbociclib on NPC cells.
We then subjected the three NPC cell lines to RNA sequencing to identify the changes in gene expression in response to palbociclib treatment for 24 hours. The differentially expressed genes were subjected to a KEGG database analysis (Fig. 1G), which revealed the downregulated expression of genes related to cell cycle progression in all three tested cell lines (Table 1). The RNA expression profiling analysis further confirmed that palbociclib induces cell-cycle arrest in NPC cells.
Oral administration of palbociclib suppressed the growth of multiple NPC xenograft models in vivo
This study included a comprehensive evaluation of the efficacy of palbociclib in six NPC preclinical xenografts models representing early- and advanced-stage NPC. Xeno32, Xeno76, and C666-1 xenografts were established from primary NPCs, Xeno23 and NPC43 were established from recurrent NPC and C17 was established from a metastatic NPC. Immunohistochemical analysis was used to evaluate the expression of p16, RB, and cyclin D1 proteins in these xenografts grown in NOD/SCID mice (Supplementary Fig. S4). Notably, all six xenografts lacked p16 protein but expressed readily detectable RB and cyclin D1.
For the treatment experiment, palbociclib was suspended in deionized water and administered daily (75 mg/kg/day) to mice bearing the Xeno32, Xeno76, C666-1, Xeno23, NPC43, and C17 xenografts for 15, 23, 13, 54, 29, and 19 days, respectively. The treatment duration in each group was dependent on the growth rate of each type of xenograft. Mice in the vehicle groups received an equivalent volume of deionized water on the treatment days. The tumor volumes were measured thrice weekly using a digital Vernier caliper. Mice were euthanized at the end of the treatment period, which was determined when the tumor diameter in the control group reached approximately 1 cm. As shown in Fig. 2A–B & Supplementary Fig. S5, oral palbociclib administration successfully inhibited the tumorigenic growth of all the NPC xenografts, and this finding supports the potential efficacy of palbociclib for the treatment of NPC.
We also performed immunohistochemical analysis to examine the expression of Ki-67, a commonly used marker of cancer cell proliferation, in NPC xenografts from the control and treatment groups at the end of the study (Fig. 2C–D). The method used to quantify Ki-67 staining in the sectioned tissues is illustrated in Supplementary Fig. S7. For all six NPC xenograft types, the percentages of Ki-67-expressing cells were significantly lower in the palbociclib treatment groups than in the control groups. The body weights of treated and control animals were measured throughout the treatment period, and no significant differences were observed. These results suggest that the palbociclib dosages administered to the NPC-bearing mice had no major adverse effects on general well-being (Fig. 2E).
The C666-1 cells used in this study could colonize the lungs of mice after tail vein injection. We therefore examined whether palbociclib could suppress the metastasis of C666-1 cells to the lungs in vivo. Four NOD/SCID mice each were included in the palbociclib treatment and control groups, and the mice were treated for 124 days. At the end of the study, lung tissues were dissected from the mice, fixed, and processed to examine the growth of C666-1 cells. Histological analysis of hematoxylin–eosin-stained lung tissues revealed that palbociclib effectively suppressed the colonization of C666-1 cells (Supplementary Fig. S8).
Combination treatment with palbociclib and other drugs revealed antagonistic effects with cisplatin but synergistic effects with SAHA against NPC cells
The treatment outcomes of patients may be worsened by drug resistance and the adverse effects of high-dose therapies. Combination treatment with two or more anticancer drugs that target different cellular pathways is a common strategy used to minimize treatment toxicity. We therefore examined the effects of combination treatments of palbociclib and other therapeutic agents, namely cisplatin or SAHA, on NPC cells. Cisplatin is commonly used for the clinical management of NPC, and SAHA has been evaluated in preclinical models of NPC (36). These drugs have distinct mechanisms of action: cisplatin induces DNA damage, whereas SAHA inhibits HDAC activity. To determine whether the combined use of palbociclib and cisplatin or SAHA would have antagonistic, synergistic or additive effects, NPC cells were treated in vitro with either palbociclib monotherapy or a combination of palbociclib with cisplatin or SAHA at serially concentrations. In all three tested NPC cell lines, the combined use of palbociclib plus cisplatin did not further suppress NPC cell growth. Rather, this combination did not act as effective as either cisplatin or palbociclib monotherapy in suppressing NPC growth (Fig 3A). This antagonistic interaction was not unexpected because cisplatin induces DNA damage, which could only be elicited once the cell entered the cell cycle. The inhibitory effects of palbociclib on cell cycle entry could protect cells from the cytotoxic effect of cisplatin. In contrast, we observed that treatment with SAHA significantly enhanced the ability of palbociclib to suppress NPC cell growth (Fig. 3B).
We also used the Chou–Talalay method to calculate the combination index (CI) of each drug combination as an indicator of the potential synergistic, additive, or antagonistic effects of the two reagents (37,38). CI values of < 1, 1, and > 1 imply synergistic, additive, and antagonistic effects, respectively (37, 38). The combination of palbociclib and cisplatin at concentrations of 2.5 µM each yielded CI values of 1.58, 2.54, and 1.041 for NPC43, C17, and C666-1, respectively, indicating a significant antagonistic effect of this combination in NPC cell lines (Fig. 3C). In contrast, the Chou–Talalay calculation confirmed the synergistic effect of the combination of palbociclib with SAHA at respective concentrations of 1.0 and 0.1 µM, which yielded CI values of 0.36, 0.38, and 0.6 in NPC43, C17, and C6661 cells, respectively (Fig. 3C).
Combined treatment with palbociclib and SAHA suppressed NPC xenograft growth in vivo
Based on the results of our Chou–Talalay analysis (37,38), we aimed to confirm the synergistic suppressive effect of combined treatment with palbociclib and SAHA on the growth of Xeno23, Xeno76, and C666-1 tumors in vivo. NPC xenograft-bearing mice were treated with palbociclib (75 mg/kg on alternate days) and/or SAHA (20 mg/kg on alternate days). This reduction in the palbociclib dose from a monotherapeutic dose of 75 mg/kg per day to the same dose on alternating days in the combination treatment was done to ensure that any potential additive or synergistic effect of SAHA could be observed. The average tumor volumes, growth curves, and histological appearances of all NPC xenografts subjected to monotherapies and combination therapies are shown in Fig. 4A. Significant tumor growth inhibition was observed in all mice in the combination palbociclib and SAHA treatment group, compared to both monotherapy groups, and the decreased tumor volume was particularly prominent in Xeno23 and C666-1 xenograft models. We further examined Ki-67 expression in NPC cells in the control and treatment groups as a measure of the cell proliferation status. In all three NPC xenograft models, all combination treatment groups exhibited significant decreases in Ki-67 expression relative to the control groups (Fig. 4B & Supplementary Fig. S9).
We subjected Xeno23 model mice to 18F-FDG microPET scans to examine the metabolic rates in the tumors after combination treatment, which were represented by the mean SUV (SUVmean). After normalization to the liver basic metabolic rate, the tumor metabolic rates were 0.76 and 0.09 in the vehicle and combination treatment groups, respectively (Supplementary Fig. S10). The body weights of mice in both groups were also measured throughout the treatment period, and no significant between-group differences were observed (Fig. 4C).
Autophagy-associated cell death as a mediator of enhanced cytotoxicity induced by combined treatment in NPC cells
We sought to explore some of the mechanisms underlying the enhanced death of NPC43 cells in response to palbociclib and SAHA monotherapy or combination therapy (Supplementary Fig. S11). The levels of multiple protein markers associated with the cell cycle, differentiation and apoptosis were analyzed by Western blotting. Palbociclib monotherapy inhibited cell cycle progression in treated NPC43 cells, as shown by the suppression of RB-Ser780 phosphorylation and cyclin A expression. SAHA monotherapy at the indicated doses was a much less effective suppressor of RB phosphorylation and cyclin A expression, which highlights the different modes of action of these drugs. We did not observe significant effects of palbociclib and SAHA monotherapy or combination therapy on involucrin (a squamous cell differentiation marker), cleaved-PARP, or cleaved-caspase 3 (apoptosis markers) in NPC43 cells treated with SAHA alone or in combination with palbociclib.
Reactivation of the EBV lytic cycle was reported to play a role in NPC cell apoptosis after drug treatment (36). Although we detected the lytic gene, BZLF1, at the single-cell level using RNAscope (Supplementary Fig. S12), less than 1% of the tumor cells expressed BZLF1 before or after the treatment, suggesting that EBV reactivation did not account for the enhanced cell death observed in response to combined treatment. Therefore, to explore the biological pathways that may have contributed to the enhanced cell death associated with combined palbociclib and SAHA treatment, we compared the RNA-sequencing profiles of three NPC cell lines (C666-1, NPC43, and C17) to identify differential gene expression (DEG) after palbociclib and SAHA treatment. The resulting Venn diagram revealed 914 upregulated genes shared by all three NPC cell lines after treatment with both drugs (Fig. 5A). A KEGG pathway analysis of these upregulated genes was conducted to identify the top 20 pathways based on the gene ratios related to each pathway. Interestingly, many of the upregulated genes were involved in autophagy pathways, as listed according to the CPDB (ConsensusPathDB) database (Fig. 5B). The autophagy-related genes involved in the enriched lysosome, macroautophagy, mitophagy, and autophagy pathways are listed in Table 2. We performed Western blot analysis of NPC cells treated with a combination of palbociclib and SAHA and identified increases in the LC3-II (the phosphatidylethanolamine conjugated form of LC3), which indicated increases in the cellular autophagy flux (Fig. 5C).
Elevated LC3-II may be attributable to enhanced autophagosome formation or blocked autophagic degradation. We next evaluated the effect of chloroquine (CQ), a specific inhibitor that blocks autophagic flux by decreasing autophagosome–lysosome fusion, on the LC3-II and viabilities of NPC cell lines. In a fully autophagic cell, CQ would induce an increased accumulation of LC3-II. If autophagy is blocked at the autophagic degradation step, however, CQ treatment would not further increase the level of LC3-II (39). As shown in Fig. 5D, we determined a higher level of LC3-II in C666-1 cells subjected to combination treatment (~5.8-fold increase after normalization to GAPDH expression) than in cells subjected to palbociclib (~2.9-fold increase) or SAHA monotherapy (~2-fold increase), indicating that combination therapy induced an increase in autophagic flux. In the presence of CQ, the LC3-II level increased by 7.6-fold in C666-1 cells treated with the combination of palbociclib and SAHA, compared to the control. Importantly, treatment with CQ also led to a significant increase in viability in cells treated with the combination therapy, compared to palbociclib monotherapy (50% vs. 31%) at 24 hours (Fig. 5E), and similar trends were observed at 48 and 72 hours and in C17 and NPC43 cells (Supplementary Fig. S13).
We further determined that knockdown of the autophagy-related proteins ATG5 and beclin-1 also reversed the NPC cell death induced by palbociclib monotherapy or palbociclib and SAHA combination treatment (Fig. 5F, Supplementary Fig. S14). We further visualized autophagosomes in cells transfected to express GFP-LC3-II and determined that these structures were enriched in cells treated with palbociclib alone or in combination with SAHA (Fig. 5G, Supplementary Fig. S15).
We also examined the levels of LC3-II in proteins extracted from C666-1 xenograft tumors from mice treated with a combination of palbociclib and SAHA. We observed a significant increase in the LC3-II/LC3-I turnover ratio in the combination treatment group relative to the monotherapy groups, consistent with the results of the Western blot analysis of cultured C666-1 cells (Fig. 5H). Taken together, our results suggest that autophagy plays a role in the cytotoxicity induced by combined treatment with palbociclib and SAHA.
Sensitivity of palbociclib-resistant NPC cells to cisplatin treatment
Cancer cells often develop mechanisms to resist the inhibitory effects of a particular drug after prolonged treatment. Consequently, NPC cells may eventually gain resistance to palbociclib. We conducted a preclinical investigation to determine whether palbociclib-resistant NPC cells would retain their responsiveness to other chemotherapeutic agents, such as cisplatin. To establish palbociclib-resistant (PD_R) NPC cell lines, we treated C666-1 and NPC43 cells with increasing doses of palbociclib over a period of 1.5 years. We then characterized the levels of cell cycle-related proteins in parental and resistant NPC43 cells in response to treatment with palbociclib or vehicle (Fig. 6A). The levels of RB and phospho-RB (Ser780) were decreased in NPC43 PD_R cells. Notably, resistant cells maintained the basal expression of cyclin A even when treated with 5 µM palbociclib, which was shown to inhibit cyclin A expression in parental NPC43 cells. NPC43 PD_R cells also expressed higher levels of cyclin E1, cyclin D2, and cyclin D3. Taken together, our results suggest that the resistant cells may have acquired alternate CDK4/6/cyclin D1/RB-independent pathways to maintain cell proliferation in the presence of palbociclib. The resistant lines also exhibited downregulated E-cadherin expression and upregulated N-cadherin expression, suggesting that these cells may be more prone to metastasis. A qPCR analysis also revealed elevated expression of the cancer stemness-related genes MMP2, MMP9, Nanog and SOX2 (Fig. 6B), suggesting that NPC43 PD_R cells may have acquired an increase in cancer stemness.
We conducted a colony formation assay to verify the palbociclib-resistant status of NPC43 PD_R cells (Fig. 6C). Parental NPC43 cells could no longer sustain colony formation under treatment with 0.8 µM palbociclib, whereas NPC43 PD_R cells could maintain colony formation even under treatment with a 10-fold higher concentration of palbociclib (8 µM). We also established a C666-1 PD_R cell line and determined the concentrations that would induce a 50% inhibition of colony formation (ICol50) in both the parental and PD_R NPC43 and C666-1 cell lines. Relative to their parental lines, NPC43 PD_R and C666-1 PD_R exhibited increases in the ICol50day16 values of 25-fold (from 0.065 to 1.645 µM) and 133.5-fold (from 0.03048 to 4.07 µM), respectively (Fig. 6D).
We next examined the responses of the two palbociclib-resistant NPC cell lines to treatment with cisplatin (Fig. 6E) and observed comparable IC50day5 values in both the parental and PD_R NPC43 and C666-1 lines. This observation suggests that palbociclib-resistant cells retain sensitivity to cisplatin, and therefore cisplatin could potentially be used to treat patients with palbociclib resistance.
Sensitivity of cisplatin-resistant NPC cells to palbociclib treatment
Platinum-based therapies are used as a first-line treatment for primary NPC, a salvage treatment for recurrent disease and a palliative treatment for metastasis (2,3,40). Therefore, we assessed whether cisplatin-resistant NPC cells would remain responsive to palbociclib treatment. In our analysis, two cisplatin-resistant sublines of NPC43 exhibited approximately 6-fold increases in the IC50 values relative to the parental NPC43 (IC50day2 increases from 6.8 to 48.58 and 51.31 µM; Fig. 6F). We then determined the efficacy of palbociclib in these cisplatin-resistant sublines and observed IC50day3 values of 28.61 µM versus 19.74 µM and 16.83 µM for the NPC43 parental versus the cisplatin-resistant sublines (Fig. 6G). This slight decrease in the IC50 indicated that the cisplatin-resistant NPC cells retained the parental sensitivity to palbociclib.