The expression of wild-type estrogen receptor β (ESR2/ERβ1) correlates with clinical outcome in patients with non–small cell lung cancer (NSCLC). However, the molecular mechanism that accounts for this association is currently poorly understood. ERβ1 was previously linked to chemotherapy response in patients with breast cancer and in breast cancer cells. The effect of the receptor in NSCLC cells after chemotherapy treatment, a common remedy for advanced NSCLC, has not been studied. Here, upregulation of ERβ1 increases the sensitivity of NSCLC cells to treatment with doxorubicin and etoposide. This effect was primarily observed in p53-defecient NSCLC cells. In these cells, ERβ1 either enhanced G2–M cell-cycle arrest by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling or induced apoptosis. The expression of p63 target genes that control G2–M checkpoint activation was altered by ERβ1 suggesting an ERβ1–p63 transcriptional cooperation in lung cancer cells that affects DNA damage response (DDR). These results suggest involvement of ERβ1 in the mechanism that regulates DNA damage response in NSCLC cells and support the potential predictive and therapeutic value of the receptor in clinical management of the disease.

Implications: This study demonstrating the impact of ERβ1 on chemosensitivity of NSCLC cells suggests the predictive value of the receptor for successful response of tumors to chemotherapy and the potential benefit of chemotherapy-treated patients from the use of ER ligands. Mol Cancer Res; 16(2); 233–42. ©2017 AACR.

Lung cancer is the most common cause of cancer-related deaths worldwide (1). Non–small cell lung cancer (NSCLC) comprises more than 85% of the diagnosed lung cancers and has a 5-year survival rate of 18% (2). Despite advances in methods of detection and availability of more treatment options, NSCLC is often diagnosed at an advanced stage and has poor prognosis. The standard of care for advanced NSCLC includes adjuvant chemotherapy and antineoplastic agents (3). These remedies are also indicated for patients with advanced disease and tumors with targetable genetic alterations that acquire resistance to treatment with kinase inhibitors (4). However, the efficacy of chemotherapy is limited due to intrinsic or acquired resistance (5, 6). Mechanisms that are implicated in chemotherapy resistance include drug inactivation by detoxifying enzymes or increased efflux, increased DNA damage tolerance and repair, increased resistance to apoptosis, and activation of survival pathways that counteract the effects of the drugs (5, 7). Pathways that mediate drug resistance are currently being investigated to identify biomarkers for better selecting patients that will respond to first-line chemotherapy and targets to combat resistance through novel therapies (8).

Cytotoxic agents elicit their effects through various mechanisms that result in activation of DNA damage response and induction of apoptosis. Treatment with DNA-damaging agents arrests the cells in different phases of the cell cycle in a p53-dependent or independent manner. Nonmalignant cells that normally express functional p53 protein are primarily arrested in G1 phase due to p53-mediated upregulation of p21, whereas p53-defective tumor cells activate the S- and G2–M-phase checkpoints (9). Targeted disruption of the remaining DNA damage response pathways in cancer cells with p53 deficiency increases the chemotherapy sensitivity through accumulation of DNA damage that activates the apoptotic machinery (9–11). This observation has led to therapeutic strategies that sensitize p53-deficient cancer cells to DNA-damaging agents by targeting the residual in these cells G2–M checkpoint (9).

The potential role of estrogens in lung cancer biology and therapy is currently under investigation. Despite the suggested association of estrogen signaling with clinical outcome, the exact effects of estrogen on NSCLC and the mechanism of action are still poorly understood (12). Estrogens mediate their effects in target tissues by acting on estrogen receptor (ER) subtypes ERα and ERβ (13). The expression of ERs has been associated with prognosis in NSCLC. While, the presence of ERα correlates with poor outcome (14, 15), conflicting data have been published regarding the clinical importance of ERβ in the disease. High expression of wild-type ERβ (ERβ1) has been associated with better survival among men and worse outcome in women and nuclear ERβ1 positivity predicts better response of lung adenocarcinoma to tyrosine kinase inhibitors (15–23). In contrast, high cytoplasmic ERβ1 levels define patients with worse overall survival (24). Consistent with the clinical associations, while a significant number of publications has reported proapoptotic effects of ERβ1 in prostate, breast, and colon cancer cells (25–27), other studies associated the receptor with the proliferative role of estrogens in breast cancer cells (28). Similar to other cancers, contradictory effects of ERβ have been reported on growth of NSCLC cells (29). We previously observed that upregulation of ERβ1 induces cell-cycle arrest and apoptosis by inhibiting oncogenic RAS signaling (30). ERβ has also been reported to sensitize cancer cells to chemotherapeutic agents. Upregulation of ERβ1 or its splice variant ERβ5 enhanced the efficacy of doxorubicin and cisplatin in breast cancer cells (31, 32). Consistently, high expression of ERβ1 has recently been associated with better prognosis in chemotherapy-treated ERα-negative breast cancer (33, 34). In addition, ligand-mediated activation of ERβ has been shown to sensitize malignant pleural mesothelioma cells to cisplatin and pemetrexed therapy by suppressing AKT signaling that leads to PARP-dependent proapoptotic cell death (35). These findings suggest the involvement of ERβ in the mechanisms that regulate DNA damage response in cancer cells and indicate the potential of the receptor to alter the efficacy of chemotherapy in NSCLC cells. Investigation of the effect of ERβ in chemotherapy response of NSCLC cells was the focus of the current study. Here, we show that ERβ1 increases the sensitivity of NSCLC cells to chemotherapeutic agents by inducing G2–M cell-cycle arrest and apoptosis.

Cell lines and reagents

NSCLC cancer cell lines (H1299, H661, H1944 and H358) were obtained from ATCC. They were cultured in RPMI-1640 (Invitrogen) media supplemented with 10% FBS (Sigma-Aldrich), 13.5 mmol/L d-Glucose and 50 μg/mL kanamycin at 37°C in a humidified incubator with 5% CO2. Doxorubicin, etoposide and nocodazole were purchased from Cell Signaling Technology. Cisplatin (cis-Platinum(II)diammine dichloride) and caffeine were purchased from Sigma-Aldrich. ERβ1 (Clone 14C8) and p84 antibodies were purchased from GeneTex (36). The primary antibodies against p-Chk1 (S345), p-Chk2 (T68) were from Cell Signaling Technology. β-Actin and FLAG (M2 clone) primary antibodies were obtained from Sigma-Aldrich.

Cell transfections

ERβ1 expression constructs were generated by cloning the full-length ERβ1 in the pIRESneo or pLenti6/V5 expression vectors (Clontech Laboratories) as described previously (30, 37). H1299 cells were transfected with empty pIRESneo vector or the recombinant pIRESneo-ERβ1 plasmid. H661, H1944, and H358 were infected with lentiviruses containing the empty plenti6/V5 vector or the recombinant pLenti6/V5-D-FLAG-ERβ1 plasmid as described previously (37). H1299 cells were transiently transfected twice with p63-specific siRNAs (Invitrogen), target sequences 1# 5′-ATTCCATGGTCGTGTGAGACAGAAG-3, and 2# 5′-AACTTAAGCGCCGAGTCGAGTACA-3′. A siRNA-targeting luciferase was used as a control (catalog. no. 12935-146, Invitrogen).

Cell survival assay

Control and ERβ1-expressing H1299, H358, H1944, and H661 cells were plated onto 96-well dishes in 10% FBS-containing medium at a density of 5,000 cells/well and treated with increasing concentrations of doxorubicin, etoposide or cisplatin (0–10 μmol/L) in quintuplicates. Seventy-two hours later, the surviving portion of the cells was measured by the Cell Titer-Blue cell viability assay, following the manufacturer's protocol (Promega). The IC50 values for the drugs in control and ERβ1-expressing cells were calculated using GraphPad Prism 5 after estimating the mean of three independent experiments.

RNA extraction and real-time PCR

Total RNA was isolated using Aurum Total RNA mini kit (Bio-Rad) and reverse-transcribed to cDNA using the iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was performed using the iTaq SYBR Green kit (Bio-Rad). All quantitative data were normalized to GAPDH and 36B4. Primers sequences for the real-time PCR analysis of CCNG2 and p63 were CCNG2-FW: 5-TGGACAGGTTCTTGGCTCTT-3, CCNG2-RV: 5-GATGGAATATTGCAGTCTTCTTCA-3, p63-FW: 5-AAGATGGTGCGACAAACAAG and p63-RV: 5-AGAGAGCATCGAAGGTGGAG-3.

Immunoblotting and immunoprecipitation

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.1% SDS, 0.5% deoxycholate, and 1% NP-40) containing protease (1 mmol/L EDTA, Roche protease inhibitor mixture, and 2 mmol/L PMSF) and phosphatase inhibitors (1 mmol/L NaF, 1 mmol/L Na3VO4, and Sigma phosphatase inhibitor mixture). The lysates were subjected to SDS-PAGE and the proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in TBST (0.05% Tween-20) for 3 hours at room temperature and probed with the primary antibodies overnight at 4°C. The membranes were incubated with secondary antibodies for 2 hours at room temperature. Proteins were visualized using enhanced chemiluminescence (ECL) detection reagents (Amersham Biosciences). We used ERβ1 (Clone 14C8) and anti-FLAG antibodies to detect ERβ1 expression due to concerns raised recently regarding the efficacy of some ERβ antibodies (36).

Flow cytometry

Cell-cycle progression of control and ERβ1-expressing cells after chemotherapy treatment was assessed by flow cytometry. After treatment, cells were trypsinized and fixed in 70% ice-cold ethanol overnight at 4°C. Cells were resuspended in a propidium iodide (100 μg/mL)/RNase A solution (50 μg/mL) and analyzed on a FACSAria II cell sorter (BD Biosciences). Analysis of cell-cycle data was performed using FlowJo software (Tristar Inc.).

ERβ1 sensitizes p53-defective NSCLC cells to chemotherapeutic agents

High expression of ERβ1 has been associated with better prognosis in chemotherapy-treated breast cancer patients (33). To investigate whether a similar association occurs in lung cancer, we tested the correlation between ERβ1 and survival in published Kaplan–Meier plotter datasets (38) and found that chemotherapy-treated ESR2high NSCLC patients have better clinical outcome compared with ESR2low patients (Supplementary Fig. S1). We previously showed that induction of ERβ1 expression in NSCLC cells that express RAS oncoproteins inhibits cell growth by disrupting RAS signaling (30). In this study, we investigated whether upregulation of ERβ1 sensitizes NSCLC cells to chemotherapy-induced cell death. To address this question, we stably expressed ERβ1 in NSCLC cells with different p53 status, given that p53 is critical determinant of the DNA damage response (39). We assessed cell survival in H1299 and H358 cells that are null for p53, in H661 that carry mutant p53 and H1994 and A549 cells that express wild-type p53 protein in the presence of increasing concentrations of cisplatin, etoposide, or doxorubicin that are used in treatment of NSCLC (40). Cell viability assays indicated a significant dose-dependent decrease in survival of doxorubicin and etoposide-treated p53-null H1299 and H358 cells after upregulation of ERβ1. While in absence of treatment ERβ1 decreased the survival of H1299 cells by less than 50%, in the presence of 1 μmol/L doxorubicin upregulation of the receptor caused a 75% reduction in the viability of the same cells (Fig. 1A and B). In contrast, induction of ERβ1 expression did not affect the viability of the cells that express mutant or wild-type p53 after treatment with the same chemotherapeutic drugs (Fig. 1C). The effect of ERβ1 on NSCLC cells was drug-specific as upregulation of ERβ1 did not alter the survival of any of the cell lines in the presence of cisplatin (Fig. 1D). These results suggest a role for ERβ1 in sensitizing NSCLC cells to chemotherapeutic agents in the absence of p53 expression.

Figure 1.

ERβ1 sensitizes p53-defective NSCLC cells to topoisomerase II inhibitors. A–C, Viability of control and ERβ1-expressing H1299, H358, H661 (p53-defective), and H1944 (wild-type p53) NSCLC cells following treatment with increasing concentrations of the topoisomerase II inhibitors doxorubicin or etoposide. Bar graphs represent the mean of three independent experiments with SEM (P < 0.05). D, Cell viability was assesed in control and ERβ1-expressing H1299 and H358 cells after treatment with increasing concentrations of cisplatin. Calculation of inhibitory concentration 50 (IC50) for doxorubicin, etoposide and cisplatin in control and ERβ1-expressing H1299, H358, H1944, and H661 cells was performed by GraphPad Prism 6.0. IC50 values shown in each graph represent mean of three independent experiments.

Figure 1.

ERβ1 sensitizes p53-defective NSCLC cells to topoisomerase II inhibitors. A–C, Viability of control and ERβ1-expressing H1299, H358, H661 (p53-defective), and H1944 (wild-type p53) NSCLC cells following treatment with increasing concentrations of the topoisomerase II inhibitors doxorubicin or etoposide. Bar graphs represent the mean of three independent experiments with SEM (P < 0.05). D, Cell viability was assesed in control and ERβ1-expressing H1299 and H358 cells after treatment with increasing concentrations of cisplatin. Calculation of inhibitory concentration 50 (IC50) for doxorubicin, etoposide and cisplatin in control and ERβ1-expressing H1299, H358, H1944, and H661 cells was performed by GraphPad Prism 6.0. IC50 values shown in each graph represent mean of three independent experiments.

Close modal

ERβ1 induces different cellular responses to chemotherapy in NSCLC cells

To elucidate the mechanism underlying the increased sensitivity of ERβ1-expressing NSCLC cells to DNA-damaging agents, p53-defiecient control and ERβ1-expressing H1299 and H358 cells were exposed to two different concentrations of doxorubicin and etoposide for 48 hours and analyzed for cell-cycle progression by FACS. As shown in Fig. 2A, treatment with these drugs arrested both control and ERβ1-expressing H1299 cells, due to absence of p53, in G2–M phase of the cell cycle in a concentration-dependent manner. However, the percentage of cells arrested in G2–M phase was significantly higher in ERβ1-expressing compared with the control cells suggesting that upregulation of the receptor leads to a stronger activation of the G2–M checkpoint in the presence of DNA damage (Fig. 2A). Relative to H1299 cells, induction of ERβ1 expression in H358 cells under the same treatment did not increase the number of cells that were arrested in G2–M phase. Instead, upregulation of ERβ1 enhanced apoptosis as indicated by the increased sub-G1 cell population in ERβ1-expressing cells after treatment with doxorubicin and in less extent with etoposide (Fig. 2B). Surprisingly, although upregulation of ERβ1 in H661 cells did not significantly alter their sensitivity to DNA-damaging agents (Fig. 1C), it abrogated the chemotherapy-induced G2–M-phase cell-cycle arrest as shown by the higher percentage of ERβ1-expressing cells in G1 phase compared with the control cells after treatment with doxorubicin (Fig. 2C). Taken together, these results suggest that different mechanisms mediate the effects of ERβ1 on survival of NSCLC cells in response to chemotherapy treatment.

Figure 2.

ERβ1 sensitizes NSCLC cells to chemotherapeutic agents through different mechanisms. A, Control and ERβ1-expressing H1299 cells were treated with two different concentrations of doxorubicin (top) and etoposide (bottom) for 48 hours. Cell-cycle profiles were analyzed by flow cytometry after staining the cells with propidium iodide (PI). Experiments were performed at least three times. B, Control and ERβ1-expressing H358 cells were either left untreated or treated with two different concentrations of doxorubicin (top) or etoposide (bottom). Forty-eight hours later, the cells were subjected to cell-cycle analysis by flow cytometry. C, Control and ERβ1-expressing H661 cells were treated with doxorubicin and analyzed by flow cytometry. The graphs in the left panels represent the percentage of cells in different phases of the cell cycle from three independent experiments. *,#, P < 0.05.

Figure 2.

ERβ1 sensitizes NSCLC cells to chemotherapeutic agents through different mechanisms. A, Control and ERβ1-expressing H1299 cells were treated with two different concentrations of doxorubicin (top) and etoposide (bottom) for 48 hours. Cell-cycle profiles were analyzed by flow cytometry after staining the cells with propidium iodide (PI). Experiments were performed at least three times. B, Control and ERβ1-expressing H358 cells were either left untreated or treated with two different concentrations of doxorubicin (top) or etoposide (bottom). Forty-eight hours later, the cells were subjected to cell-cycle analysis by flow cytometry. C, Control and ERβ1-expressing H661 cells were treated with doxorubicin and analyzed by flow cytometry. The graphs in the left panels represent the percentage of cells in different phases of the cell cycle from three independent experiments. *,#, P < 0.05.

Close modal

ERβ1 regulates the activity of G2–M checkpoint effectors in NSCLC cells

Upon DNA damage, activation of ataxia telangiectasia, mutated (ATM) and ataxia telangiectasia and RAD3-related protein (ATR) leads to activation of the checkpoint kinase Chk2 through phosphorylation at Thr-68 and Chk1 through phosphorylation at Ser-345 (41, 42). Activation of Chk1 and Chk2 causes G2–M-phase cell-cycle arrest (43). To identify the molecular mechanisms that are responsible for the stronger cell-cycle arrest in ERβ1-expressing NSCLC cells in presence of chemotherapeutics, control and ERβ1-expressing H1299 cells were incubated with two different concentrations of doxorubicin and etoposide for 24 and 48 hours and the phosphorylation status of Chk1 and Chk2 that indicates activation was examined. In response to treatment, ERβ1-expressing cells displayed higher levels of Chk1 and Chk2 phosphorylation compared with the control cells and the effect of the receptor appeared to be dependent on the dose of the drug and the time of treatment (Fig. 3A). These results strengthen our findings from the cell-cycle analysis showing that ERβ1 induces G2–M arrest in response to DNA damage.

Figure 3.

ERβ1 alters DNA damage response pathways in response to chemotherapeutic agents. A and B, Levels of phospho (p)-Chk1(S345), p-Chk2(T68) and ERβ1 in control and ERβ1-expressing H1299 (A) and H358 (B) cells after treatment with two different concentrations of doxorubicin (left) and etoposide (right) for 24 and 48 hours. FLAG-ERβ1 in H358 cells was detected with anti-FLAG antibody. Of note, ERβ1 expression decreases with increasing concentrations of etoposide. C, Expression of p-Chk1(S345) and p-Chk2(T68) in control and ERβ1-expressing H661 cells following treatment with doxorubicin for 24 and 48 hours.

Figure 3.

ERβ1 alters DNA damage response pathways in response to chemotherapeutic agents. A and B, Levels of phospho (p)-Chk1(S345), p-Chk2(T68) and ERβ1 in control and ERβ1-expressing H1299 (A) and H358 (B) cells after treatment with two different concentrations of doxorubicin (left) and etoposide (right) for 24 and 48 hours. FLAG-ERβ1 in H358 cells was detected with anti-FLAG antibody. Of note, ERβ1 expression decreases with increasing concentrations of etoposide. C, Expression of p-Chk1(S345) and p-Chk2(T68) in control and ERβ1-expressing H661 cells following treatment with doxorubicin for 24 and 48 hours.

Close modal

In contrast to H1299 cells, the phosphorylation of Chk1 was higher in control compared with ERβ1-expressing H358 and H661 cells after treatment with doxorubicin or etoposide (H358) for 24 and 48 hours suggesting that ERβ1 rather inhibits the G2–M cell-cycle checkpoint in these cells (Fig. 3B and C). The inhibition of this specific DNA damage response pathway upon ERβ1 upregulation is consistent with the abrogation of the G2–M cell-cycle arrest that was detected in ERβ1-expressing H358 and H661 cells and may account for the enhanced apoptosis that was observed in ERβ1-expressing H358 cells in cell-cycle analysis (Fig. 2B and C). Taken together, these results suggest that in response to DNA damage, ERβ1 differentially regulates the G2–M cell cycle checkpoint in different p53-defective NSCLC cells.

ERβ1 delays G2–M progression of NSCLC cells in response to chemotherapy

To corroborate that ERβ1 regulates the G2–M cell cycle checkpoint in chemotherapy-treated NSCLC cells, we monitored cell-cycle progression in control and ERβ1-expressing H1299 cells following treatment with doxorubicin in the presence of nocodazole. Given that nocodazole arrests the cells in G2–M phase, we examined whether ERβ1 delays the transition from G2–M to G1 phase after the release of the cells from nocodazole block (44). Following treatment for 18 hours with nocodazole, cells were released and treated for an additional 6 hours with doxorubicin. As seen in Fig. 4A, 6 hours after release from nocodazole block, a significantly higher percentage of ERβ1-expressing cells remained in G2–M phase of the cell cycle compared with the control cells.

Figure 4.

ERβ1 delays G2–M progression in NSCLC cells in response to chemotherapy. A, Control and ERβ1-expressing H1299 cells were treated with doxorubicin in the presence of nocodazole (15 μg/mL) for 18 hours to induce G2–M arrest. Cells were incubated in media containing doxorubicin for another 6 hours and the cell cycle was analyzed at 0 and 6 hours after nocodazole release. B, Immunoblot analysis of p-Chk1(S345) following treatment of control and ERβ1-expressing H1299 cells with two different concentrations of doxorubicin in the presence of caffeine for 48 hours. β-Actin was used as loading control. C, Cell-cycle analysis of ERβ1-expressing H1299 cells upon treatment with doxorubicin in the presence of caffeine for 48 hours. Bar graphs on the right represent quantitative data of the percentage of cells in different phases of the cell cycle from three independent experiments. *,#, P < 0.05.

Figure 4.

ERβ1 delays G2–M progression in NSCLC cells in response to chemotherapy. A, Control and ERβ1-expressing H1299 cells were treated with doxorubicin in the presence of nocodazole (15 μg/mL) for 18 hours to induce G2–M arrest. Cells were incubated in media containing doxorubicin for another 6 hours and the cell cycle was analyzed at 0 and 6 hours after nocodazole release. B, Immunoblot analysis of p-Chk1(S345) following treatment of control and ERβ1-expressing H1299 cells with two different concentrations of doxorubicin in the presence of caffeine for 48 hours. β-Actin was used as loading control. C, Cell-cycle analysis of ERβ1-expressing H1299 cells upon treatment with doxorubicin in the presence of caffeine for 48 hours. Bar graphs on the right represent quantitative data of the percentage of cells in different phases of the cell cycle from three independent experiments. *,#, P < 0.05.

Close modal

Moreover, to strengthen our findings showing that ERβ1 prolongs G2–M phase arrest by acting on the pathway that activates Chk1, we assessed control and ERβ1-expressing H1299 cells after treatment with caffeine, a potent ATM/ATR inhibitor in the presence of doxorubicin (45). As shown in Fig. 4B and C, treatment with caffeine reversed the ERβ1-induced phosphorylation of Chk1 and the delay of the cells into G2–M phase, strengthening our initial hypothesis that in p53-deficient NSCLC cells, the receptor regulates the DNA damage response pathways that control the G2–M checkpoint. Taken together, these results strongly suggest that in response to DNA damage, ERβ1 leads to a prolonged G2–M checkpoint activation in NSCLC cells by regulating the induction of the DNA damage response pathway.

ERβ1 synergizes with p63 to enhance the chemosensitivity of NSCLC cells

One of the mechanisms by which estrogen receptors elicit their tumor-associated functions involves their transcriptional cooperation with the p53-family proteins p53 and p63 (46, 47). To further elucidate the molecular pathways that mediate the effect of ERβ1 on chemotherapy response of NSCLC cells, we investigated whether ERβ1 acts through p63 in H1299 cells. These p53-null cells express high levels of p63 and, thus, rely on this gene to activate the DNA damage response (Supplementary Fig. S2). As a transcription factor, p63 affects the expression of several genes that regulate cell-cycle progression including Cyclin G2 (CCNG2) that activates the G2–M cell-cycle checkpoint (48). It was previously shown that CCNG2 is upregulated by ERβ1 (46) and repressed by ERα and estrogen in cancer cells (49). We initially examined whether disruption of p63 expression alters the effect of ERβ1 on chemotherapy response of H1299 cells. Indeed, downregulation of p63 in ERβ1-expressing H1299 cells reversed the ERβ1-mediated G2–M-phase cell-cycle arrest (Fig. 5A) and phosphorylation of Chk1 and Chk2 (Fig. 5B). In addition, in response to doxorubicin treatment, knockdown of p63 in the same cells reversed the upregulation of CCNG2 that seems to be essential for the ERβ1-mediated G2–M-phase cell-cycle arrest (Fig. 5C). These results demonstrate the importance of p63 and its downstream signaling in mediating the effect of ERβ1 on chemotherapy response of H1299 cells (Fig. 5D).

Figure 5.

ERβ1 synergizes with p63 to enhance the chemosensitivity of NSCLC cells. A, ERβ1-expressing H1299 NSCLC cells were transfected twice with scramble or siRNA against p63 (sip63). Forty-eight hours later, the cells were treated with doxorubicin for an additional 48 hours and their cell cycle was analyzed by flow cytometry. B, Levels of p-Chk1(S345) and p-Chk2(T68) in ERβ1-expressing H1299 cells after transfection with scramble or siRNA against p63 and treatment with doxorubicin for 48 hours. C, mRNA expression of CCNG2 in ERβ1-expressing H1299 cells after transfection with scramble or siRNA against p63 and treatment with doxorubicin for 48 hours. D, Schematic representation of the mechanism employed by ERβ1 to induce G2–M cell-cycle arrest in response to chemotherapy treatment in NSCLC cells. ERβ1 increases the activity of Chk1 and Chk2 that prolongs the chemotherapy-induced G2–M cell-cycle arrest. *,#, P < 0.05.

Figure 5.

ERβ1 synergizes with p63 to enhance the chemosensitivity of NSCLC cells. A, ERβ1-expressing H1299 NSCLC cells were transfected twice with scramble or siRNA against p63 (sip63). Forty-eight hours later, the cells were treated with doxorubicin for an additional 48 hours and their cell cycle was analyzed by flow cytometry. B, Levels of p-Chk1(S345) and p-Chk2(T68) in ERβ1-expressing H1299 cells after transfection with scramble or siRNA against p63 and treatment with doxorubicin for 48 hours. C, mRNA expression of CCNG2 in ERβ1-expressing H1299 cells after transfection with scramble or siRNA against p63 and treatment with doxorubicin for 48 hours. D, Schematic representation of the mechanism employed by ERβ1 to induce G2–M cell-cycle arrest in response to chemotherapy treatment in NSCLC cells. ERβ1 increases the activity of Chk1 and Chk2 that prolongs the chemotherapy-induced G2–M cell-cycle arrest. *,#, P < 0.05.

Close modal

Chemotherapy remains the standard of care in management of advanced NSCLC. However, disease progression and treatment–related toxicities continue to limit survival. Targeted therapy is currently being investigated with the aim to improve outcomes with less toxic effects (50). In addition to identifying activating mutations in targetable oncogenes including EGFR and ALK (51), recent studies have reported the expression of estrogen receptors in NSCLC and implicated estrogen signaling in the development and progression of the disease. The presence of ERs in lung tumors indicates that targetable signaling is no longer restricted to receptor tyrosine kinases and their downstream effectors with the real possibility of utilizing ERs as complementary prognostic markers and therapeutic targets (52). ERs have been associated with outcomes in patients with NSCLC and the regulation of survival in NSCLC cells. We previously observed inhibition of cell-cycle progression and induction of apoptosis following upregulation of ERβ1 in NSCLC cells with activating RAS mutations (30). Lung adenocarcinomas with undruggable RAS oncoproteins belong to a large NSCLC entity that is still treated with conventional chemotherapy (51). In this study, we examined whether ERβ1 increases the efficacy of chemotherapeutic agents in NSCLC cells.

Our experiments showed decreased cell survival in doxorubicin and etoposide-treated NSCLC cells after upregulation of ERβ1. In contrast, expression of ERβ1 did not alter the efficacy of cisplatin suggesting that ERβ1 acts as chemosensitizer in a drug-specific manner and its effect may be associated with the mechanism of drug action. Treatment with doxorubicin and etoposide primarily induces G2–M cell-cycle arrest by inhibiting topoisomerase II activity (53) and further enhancement or abrogation of this specific cell-cycle arrest is likely to promote cell death (43). Our cell-cycle analysis shows that ERβ1 alters the activity of G2–M-phase checkpoint in doxorubicin- and etoposide-treated NSCLC cells but not in cells treated with cisplatin that, as an intercalating agent, stalls the cells largely in S-phase of the cell cycle (54). Despite the common use of cisplatin and etoposide in treatment of NSCLC, chemotherapy or chemoradiation therapy is complex and the agents needed to achieve optimal outcomes are currently not well defined (55). The chemosensitizing effect of ERβ1 in doxorubicin-treated NSCLC cells may indicate the ability of the receptor to increase the therapeutic index of agents that do not represent standard treatment. In addition to the drug-specific effect, ERβ1 seems to regulate chemotherapy sensitivity in cell context–dependent manner. We found that ERβ1 decreases the viability of chemotherapy-treated NSCLC cells that lack p53 tumor suppressor function. The absence of functional p53 protein alters the DNA damage response of cancer cells and strategies have been developed to target p53-defective tumors (56). For example, alteration of the increased cell-cycle checkpoint function in G2 phase that occurs preferentially in p53-defective cancer cells has been suggested to sensitize cancer cells to DNA-damaging agents (57). Thus, the amendment of the G2–M-phase checkpoint that was observed in ERβ1-expressing NSCLC cells may account for their decreased survival following treatment with chemotherapy.

Further analysis of cell-cycle progression revealed two distinct mechanisms of ERβ1 action. In H1299 cells, ERβ1 prolongs the chemotherapy-induced G2–M cell-cycle arrest by increasing the activity of the G2–M cell-cycle checkpoint inducers Chk1 and Chk2. This phenotype seems to rely on activation of the DNA damage sensors ATM and/or ATR, as inhibition of the pathway at this early stage diminishes the effect of the receptor. On the other hand, ERβ1 decreased the viability of chemotherapy-treated H358 cells by inducing apoptosis. H358 cells are more sensitive to doxorubicin than H1299 cells and this may account for the specific action of ERβ1 in these cells (58). Upon chemotherapy treatment, the active levels of Chk1 were lower in ERβ1-expressing compared with the control H358 cells, suggesting that the receptor abrogates the drug-induced G2–M cell-cycle checkpoint. The inhibition of the G2–M checkpoint under DNA damage may explain the chemosensitizing effect of ERβ1 in specific type of NSCLC cells (9).

The effect of ERβ1 on ATM/ATR in H1299 cells implies that the receptor operates at an early stage during the DNA damage response to induce G2–M cell-cycle arrest. It is also evident that ERβ1 regulates p53-independent DNA damage response pathways in NSCLC cells. In absence of p53, other members of the p53 family of transcription factors such as p63 and p73 function to activate the DNA damage response (59) and this may require their increased expression in p53-deficient cells (60). Because of its differential expression in NSCLC cells in which ERβ1 elicited distinct actions, p63 was considered as a potential ERβ1-interacting factor. Knockdown of p63 reversed the effect of ERβ1 on cell cycle of chemotherapy-treated NSCLC cells, indicating its involvement in the mechanism of ERβ1 action. Moreover, we previously showed that ERβ1 interacts with p63 to promote upregulation of p63 target genes in breast cancer cells (46). Among these, Cyclin G2 (CCNG2) is an estrogen-regulated gene that is activated by p63 to control the G2–M cell-cycle checkpoint (61, 62). We detected upregulation of CCNG2 upon DNA damage in ERβ1-expressing cells which strengthens our hypothesis that ERβ1 influences chemotherapy response in NSCLC cells by regulating major DNA damage response pathways (63).

In this study, we suggest that ERβ1 affects the chemosensitivity of NSCLC cells and this may correlate with its ability to regulate DNA damage response pathways. The increased cytotoxic effect of chemotherapeutics in NSCLC cells that lack p53 in the presence of ERβ1 suggests that chemotherapy-treated patients whose p53-defective lung tumors are positive for the receptor may benefit from treatment with ER ligands. This effect may also explain the previously reported association of ERβ1 with better survival in patients with NSCLC (16, 18). Given the expression of the receptor in other tumors, its ability to alter chemotherapy response may impact on other types of chemotherapy-treated cancers. As a support of this, high expression of ERβ1 has recently been associated with better prognosis in chemotherapy-treated patients with ERα-negative breast cancer (33, 34). Thus, assessing the expression of ERβ in cancer patients including those with NSCLC might hold a predictive value for their successful response to chemotherapy. Further understanding of the transcriptional activity of ERβ on target genes that regulate DNA damage response in lung cancer cells should establish ERβ as an important regulator of chemotherapy response in lung cancer.

No potential conflicts of interest were disclosed.

Conception and design: F. Nikolos, C. Thomas

Development of methodology: F. Nikolos, C. Thomas

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): F. Nikolos, I. Bado, J.A. Gustafsson

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Nikolos, C. Thomas, I. Bado, J.A. Gustafsson

Writing, review, and/or revision of the manuscript: F. Nikolos, C. Thomas, J.A. Gustafsson

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Thomas

Study supervision: C. Thomas, J.A. Gustafsson

J.A. Gustafsson was supported by the Robert A. Welch Foundation (E-0004). This work was supported by the Swedish Cancer Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2016
.
CA Cancer J Clin
2016
;
66
:
7
30
.
2.
Miller
KD
,
Siegel
RL
,
Lin
CC
,
Mariotto
AB
,
Kramer
JL
,
Rowland
JH
, et al
Cancer treatment and survivorship statistics, 2016
.
CA Cancer J Clin
2016
;
66
:
271
89
.
3.
Bareschino
MA
,
Schettino
C
,
Rossi
A
,
Maione
P
,
Sacco
PC
,
Zeppa
R
, et al
Treatment of advanced non small cell lung cancer
.
J Thorac Dis
2011
;
3
:
122
33
.
4.
Mok
TS
,
Wu
YL
,
Thongprasert
S
,
Yang
CH
,
Chu
DT
,
Saijo
N
, et al
Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma
.
N Engl J Med
2009
;
361
:
947
57
.
5.
Kim
ES
. 
Chemotherapy resistance in lung cancer
.
Adv Exp Med Biol
2016
;
893
:
189
209
.
6.
Dong
X
,
Xu
C
,
Lei
P
,
Zhang
W
. 
The new concepts on overcoming drug resistance in lung cancer
.
Drug Des Devel Ther
2014
;
8
:
735
.
7.
Almeida
GM
,
Duarte
TL
,
Farmer
PB
,
Steward
WP
,
Jones
GD
. 
Multiple end-point analysis reveals cisplatin damage tolerance to be a chemoresistance mechanism in a NSCLC model: Implications for predictive testing
.
Int J Cancer
2008
;
122
:
1810
9
.
8.
Lwin
Z
,
Riess
JW
,
Gandara
D
. 
The continuing role of chemotherapy for advanced non-small cell lung cancer in the targeted therapy era
.
J Thorac Dis
2013
;
5
Suppl 5
:
S556
64
.
9.
Dixon
H
,
Norbury
CJ
. 
Therapeutic exploitation of checkpoint defects in cancer cells lacking p53 function
.
Cell Cycle
2002
;
1
:
362
8
.
10.
Vogelstein
B
,
Lane
D
,
Levine
AJ
. 
Surfing the p53 network
.
Nature
2000
;
408
:
307
10
.
11.
Vousden
KH
,
Lu
X
. 
Live or let die: the cell's response to p53
.
Nat Rev Cancer
2002
;
2
:
594
604
.
12.
Kazmi
N
,
Márquez-Garbán
DC
,
Aivazyan
L
,
Hamilton
N
,
Garon
EB
,
Goodglick
L
, et al
The role of estrogen, progesterone and aromatase in human non-small-cell lung cancer
.
Lung Cancer Manag
2012
;
1
:
259
72
.
13.
Thomas
C
,
Gustafsson
. 
The different roles of ER subtypes in cancer biology and therapy
.
Nat Rev Cancer
2011
;
11
:
597
608
.
14.
Kawai
H
,
Ishii
A
,
Washiya
K
,
Konno
T
,
Kon
H
,
Yamaya
C
, et al
Combined overexpression of EGFR and estrogen receptor alpha correlates with a poor outcome in lung cancer
.
Anticancer Res
2005
;
25
:
4693
8
.
15.
Rades
D
,
Setter
C
,
Dahl
O
,
Schild
SE
,
Noack
F
. 
The prognostic impact of tumor cell expression of estrogen receptor-α, progesterone receptor, and androgen receptor in patients irradiated for nonsmall cell lung cancer
.
Cancer
2012
;
118
:
157
63
.
16.
Kawai
H
,
Ishii
A
,
Washiya
K
,
Konno
T
,
Kon
H
,
Yamaya
C
, et al
Human cancer biology estrogen receptor A and B are prognostic factors in non-small cell lung cancer
.
Clin Cancer Res
2005
;
11
:
5084
9
.
17.
Nose
N
,
Sugio
K
,
Oyama
T
,
Nozoe
T
,
Uramoto
H
,
Iwata
T
, et al
Association between estrogen receptor-beta expression and epidermal growth factor receptor mutation in the postoperative prognosis of adenocarcinoma of the lung
.
J Clin Oncol
2009
;
27
:
411
7
.
18.
Nose
N
,
Uramoto
H
,
Iwata
T
,
Hanagiri
T
,
Yasumoto
K
. 
Expression of estrogen receptor beta predicts a clinical response and longer progression-free survival after treatment with EGFR-TKI for adenocarcinoma of the lung
.
Lung Cancer
2011
;
71
:
350
5
.
19.
Guldhammer
B
,
Fischer
BM
,
Pappot
H
,
Skov
BG
. 
Oestrogen receptor beta over expression in males with non-small cell lung cancer is associated with better survival
.
Lung Cancer
2008
;
59
:
88
94
.
20.
Wang
Z
,
Li
Z
,
Ding
X
,
Shen
Z
,
Liu
Z
,
An
T
, et al
ERβ localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations
.
Sci Rep
2015
;
5
:
11392
.
21.
Luo
Z
,
Wu
R
,
Jiang
Y
,
Qiu
Z
,
Chen
W
,
Li
W
. 
Overexpression of estrogen receptor beta is a prognostic marker in non-small cell lung cancer: a meta-analysis
.
Int J Clin Exp Med
2015
;
8
:
8686
97
.
22.
Verma
MK
,
Miki
Y
,
Sasano
H
. 
Sex steroid receptors in human lung diseases
.
J Steroid Biochem Mol Biol
2011
;
127
:
216
22
.
23.
Mah
V
,
Marquez
D
,
Alavi
M
,
Maresh
EL
,
Zhang
L
,
Yoon
N
, et al
Expression levels of estrogen receptor beta in conjunction with aromatase predict survival in non-small cell lung cancer
.
Lung Cancer
2011
;
74
:
318
25
.
24.
Stabile
LP
,
Dacic
S
,
Land
SR
,
Lenzner
DE
,
Dhir
R
,
Acquafondata
M
, et al
Combined analysis of estrogen receptor beta-1 and progesterone receptor expression identifies lung cancer patients with poor outcome
.
Clin Cancer Res
2011
;
17
:
154
64
.
25.
Rajapaksa
G
,
Nikolos
F
,
Bado
I
,
Clarke
R
,
Gustafsson
,
Thomas
C
. 
ERβ decreases breast cancer cell survival by regulating the IRE1/XBP-1 pathway
.
Oncogene
2015
;
34
:
4130
41
.
26.
Hartman
J
,
Edvardsson
K
,
Lindberg
K
,
Zhao
C
,
Williams
C
,
Ström
A
, et al
Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells
.
Cancer Res
2009
;
69
:
6100
6
.
27.
Dey
P
,
Jonsson
P
,
Hartman
J
,
Williams
C
,
Ström
A
,
Gustafsson
. 
Estrogen receptors β1 and β2 have opposing roles in regulating proliferation and bone metastasis genes in the prostate cancer cell line PC3
.
Mol Endocrinol
2012
;
26
:
1991
2003
.
28.
Ma
R
,
Karthik
GM
,
Lövrot
J
,
Haglund
F
,
Rosin
G
,
Katchy
A
, et al
Estrogen receptor β as a therapeutic target in breast cancer stem cells
.
J Natl Cancer Inst
2017
;
109
:
1
14
.
29.
Siegfried
JM
,
Hershberger
PA
,
Stabile
LP
. 
Estrogen receptor signaling in lung cancer
.
Semin Oncol
2009
;
36
:
524
31
.
30.
Nikolos
F
,
Thomas
C
,
Rajapaksa
G
,
Bado
I
,
Gustafsson
JA
. 
ER regulates NSCLC phenotypes by controlling oncogenic RAS signaling
.
Mol Cancer Res
2014
;
12
:
843
54
.
31.
Thomas
CG
,
Strom
A
,
Lindberg
K
,
Gustafsson
JA
. 
Estrogen receptor beta decreases survival of p53-defective cancer cells after DNA damage by impairing G2/M checkpoint signaling
.
Breast Cancer Res Treat
2011
;
127
:
417
27
.
32.
Lee
MT
,
Ho
SM
,
Tarapore
P
,
Chung
I
,
Leung
YK
. 
Estrogen receptor β isoform 5 confers sensitivity of breast cancer cell lines to chemotherapeutic agent-induced apoptosis through interaction with Bcl2L12
.
Neoplasia
2013
;
15
:
1262
71
.
33.
Elebro
K
,
Borgquist
S
,
Rosendahl
AH
,
Markkula
A
,
Simonsson
M
,
Jirström
K
, et al
High estrogen receptor b expression is prognostic among adjuvant chemotherapy– treated patients—results from a population-based breast cancer cohort
.
Clin Cancer Res
2017
;
23
:
766
77
.
34.
Wang
J
,
Zhang
C
,
Chen
K
,
Tang
H
,
Tang
J
,
Song
C
, et al
ERβ1 inversely correlates with PTEN/PI3K/AKT pathway and predicts a favorable prognosis in triple-negative breast cancer
.
Breast Cancer Res Treat
2015
;
152
:
255
69
.
35.
Pinton
G
,
Manente
AG
,
Daga
A
,
Cilli
M
,
Rinaldi
M
,
Nilsson
S
, et al
Agonist activation of estrogen receptor beta (ERβ) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity
.
Mol Cancer
2014
;
13
:
227
.
36.
Nelson
AW
,
Groen
AJ
,
Miller
JL
,
Warren
AY
,
Holmes
KA
,
Tarulli
GA
, et al
Comprehensive assessment of estrogen receptor beta antibodies in cancer cell line models and tissue reveals critical limitations in reagent specificity
.
Mol Cell Endocrinol
2017
;
440
:
138
50
.
37.
Thomas
C
,
Rajapaksa
G
,
Nikolos
F
,
Hao
R
,
Katchy
A
,
McCollum
CW
, et al
ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR
.
Breast Cancer Res
2012
;
14
:
R148
.
38.
Zhu
CQ
,
Ding
K
,
Strumpf
D
,
Weir
BA
,
Meyerson
M
,
Pennell
N
, et al
Prognostic and predictive gene signature for adjuvant chemotherapy in resected non–small-cell lung cancer
.
J Clin Oncol
2010
;
28
:
4417
24
.
39.
Jackson
JG
,
Pant
V
,
Li
Q
,
Chang
LL
,
Quintás-Cardama
A
,
Garza
D
, et al
P53-mediated senescence impairs the apoptotic response to chemotherapy and clinical outcome in breast cancer
.
Cancer Cell
2012
;
21
:
793
806
.
40.
Cosaert
J
,
Quoix
E
. 
Platinum drugs in the treatment of non-small-cell lung cancer
.
Br J Cancer
2002
;
87
:
825
33
.
41.
Kuntz
K
,
O'Connell
MJ
. 
The G(2) DNA damage checkpoint: could this ancient regulator be the Achilles heel of cancer?
Cancer Biol Ther
2009
;
8
:
1433
9
.
42.
Zhou
BB
,
Elledge
SJ
. 
The DNA damage response: putting checkpoints in perspective
.
Nature
2000
;
408
:
433
9
.
43.
Taylor
WR
,
Stark
GR
. 
Regulation of the G2/M transition by p53
.
Oncogene
2001
;
20
:
1803
15
.
44.
Vasquez
RJ
,
Howell
B
,
Yvon
AM
,
Wadsworth
P
,
Cassimeris
L
. 
Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro
.
Mol Biol Cell
1997
;
8
:
973
85
.
45.
Sarkaria
JN
,
Busby
EC
,
Tibbetts
RS
,
Roos
P
,
Taya
Y
,
Karnitz
LM
, et al
Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine
.
Cancer Res
1999
;
59
:
4375
82
.
46.
Bado
I
,
Nikolos
F
,
Rajapaksa
G
,
Gustafsson
,
Thomas
C
. 
ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function
.
Oncotarget
2016
;
7
:
13599
611
.
47.
Berger
C
,
Qian
Y
,
Chen
X
. 
The p53-estrogen receptor loop in cancer
.
Curr Mol Med
2013
;
13
:
1229
40
.
48.
Dohn
M
,
Zhang
S
,
Chen
X
. 
p63α and ΔNp63α can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes
.
Oncogene
2001
;
20
:
3193
205
.
49.
Stossi
F
,
Madak-Erdogan
Z
,
Katzenellenbogen
BS
. 
Estrogen receptor alpha represses transcription of early target genes via p300 and CtBP1
.
Mol Cell Biol
2009
;
29
:
1749
59
.
50.
Yoon
SM
,
Shaikh
T
,
Hallman
M
. 
Therapeutic management options for stage III non-small cell lung cancer
.
World J Clin Oncol
2017
;
8
:
1
20
.
51.
Collisson
EA
,
Campbell
JD
,
Brooks
AN
,
Berger
AH
,
Lee
W
,
Chmielecki
J
, et al
Comprehensive molecular profiling of lung adenocarcinoma
.
Nature
2014
;
511
:
543
50
.
52.
Niikawa
H
,
Suzuki
T
,
Miki
Y
,
Suzuki
S
,
Nagasaki
S
,
Akahira
J
, et al
Intratumoral estrogens and estrogen receptors in human non-small cell lung carcinoma
.
Clin Cancer Res
2008
;
14
:
4417
26
.
53.
Nitiss
JL
. 
Targeting DNA topoisomerase II in cancer chemotherapy
.
Nat Rev Cancer
2009
;
9
:
338
50
.
54.
Dasari
S
,
Tchounwou
PB
. 
Cisplatin in cancer therapy: molecular mechanisms of action
.
Eur J Pharmacol
2014
;
740
:
364
78
.
55.
Wang
L
,
Wu
S
,
Ou
G
,
Bi
N
,
Li
W
,
Ren
H
, et al
Randomized phase II study of concurrent cisplatin/etoposide or paclitaxel/carboplatin and thoracic radiotherapy in patients with stage III non-small cell lung cancer
.
Lung Cancer
2012
;
77
:
89
96
.
56.
Reaper
PM
,
Griffiths
MR
,
Long
JM
,
Charrier
JD
,
MacCormick
S
,
Charlton
PA
, et al
Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR
.
Nat Chem Biol
2011
;
7
:
428
30
.
57.
Wang
Q
,
Fan
S
,
Eastman
A
,
Worland
PJ
,
Sausville
EA
,
O'Connor
PM
. 
UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53
.
J Natl Cancer Inst
1996
;
88
:
956
65
58.
Lv
Y
,
Yanan
H
,
Yu
X
,
Liu
R
,
Zhang
S
,
Zheng
X
, et al
TopBP1 contributes to the chemoresistance in non-small cell lung cancer through upregulation of p53
.
Drug Des Devel Ther
2016
;
10
:
3053
64
.
59.
Lin
YL
,
Sengupta
S
,
Gurdziel
K
,
Bell
GW
,
Jacks
T
,
Flores
ER
. 
p63 and p73 transcriptionally regulate genes involved in DNA repair
.
PLoS Genet
2009
;
5
:
e1000680
.
60.
Suliman
Y
,
Opitz
OG
,
Avadhani
A
,
Burns
TC
,
El-Deiry
W
,
Wong
DT
, et al
p63 expression is associated with p53 loss in oral-esophageal epithelia of p53-deficient mice
.
Cancer Res
2001
;
61
:
6467
73
.
61.
Lin
CY
,
Ström
A
,
Vega
VB
,
Kong
SL
,
Yeo
AL
,
Thomsen
JS
, et al
Discovery of estrogen receptor α target genes and response elements in breast tumor cells
.
Genome Biol
2004
;
5
:
R66
.
62.
Adorno
M
,
Cordenonsi
M
,
Montagner
M
,
Dupont
S
,
Wong
C
,
Hann
B
, et al
A mutant-p53/Smad complex opposes p63 to empower TGFβ-induced metastasis
.
Cell
2009
;
137
:
87
98
.
63.
Zimmermann
M
,
Arachchige-Don
AS
,
Donaldson
MS
,
Dallapiazza
RF
,
Cowan
CE
,
Horne
MC
. 
Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin
.
J Biol Chem
2012
;
287
:
22838
53
.