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Overexpression of epidermal growth factor receptor (EGFR) is found in over 80% of head and neck squamous cell carcinomas (HNSCC) and associated with poor clinical outcomes. EFGR selective tyrosine kinase inhibitors (TKIs) or antibodies have recently emerged as promising treatments for solid tumors, including HNSCC, though the response rate to these agents is low. p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, is required for apoptosis induced by p53 and various chemotherapeutic agents. In this study, we show that PUMA induction is correlated with EGFR-TKI sensitivity, and is mediated through the p53 family protein p73β and inhibition of the PI3K/AKT pathway. In some HNSCC cells, the gefitinib-induced degradation of oncogenic ΔNp63 seems to facilitate p73-mediated PUMA transcription. Inhibiting PUMA expression by small hairpin RNA (shRNA) impairs gefitinib-induced apoptosis. Furthermore, PUMA or BH3 mimetics sensitize HNSCC cells to gefitinib-induced apoptosis. Our results suggest that PUMA induction through p73 represents a new mechanism of EGFR inhibitor-induced apoptosis, and provide potential ways for enhancing and predicting the sensitivity to EGFR-targeted therapies in HNSCC.
Epidermal growth factor receptor (EGFR, erbB1) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormal EGFR signaling is common in a wide range of cancers (Citri and Yarden, 2006; Kalyankrishna and Grandis, 2006). Head and neck cancer, over 95% of which are squamous cell carcinoma (HNSCC), is the eighth most common cancer worldwide (Jemal et al., 2007). Overexpression of EGFR has been detected in >80% of HNSCC and is a marker of poor prognosis (Kalyankrishna and Grandis, 2006). EGFR has therefore become a rational therapeutic target for the treatment of HNSCC among other malignancies (Kalyankrishna and Grandis, 2006). EGFR is activated through receptor dimerization and phosphorylation on multiple tyrosine residues. The downstream intracellular targets of EGFR signaling include Ras/MAP kinase, phophotidylinositol-3-kinase (PI3K), phospholipase-Cγ (PLC-γ), protein kinase C (PKC), and signal transducer and activator of transcription (STATs) (Citri and Yarden, 2006).
EGFR-targeting agents currently in clinical use include selective tyrosine kinase inhibitors (TKIs), such as gefitinib (ZD1839/Iressa) and erlotinib (OSI774/Tarceva), and the monoclonal antibodies cetuximab (C225/Eribitux) and panitumimab (Vectivix). These agents have been shown to induce growth suppression, apoptosis, and/or chemo- and radiosensitization in HNSCC cells and xenograft tumors (Kalyankrishna and Grandis, 2006). Cetuximab combined with radiation has recently been shown to improve locoregional control and reduce mortality (Bonner et al., 2006), and was approved by FDA as the first new treatment in the last 45 years for HNSCC patients (Kalyankrishna and Grandis, 2006). Despite encouraging developments, EGFR-targeted therapies only work in a relatively small percentage (10–20%) of cancer patients (Bonner et al., 2006; Choong and Cohen, 2006). The critical intracellular molecular targets and the mechanism underlying variable responses to these therapies remain elusive.
The BH3-only protein PUMA (p53 upregulated modulator of apoptosis), was initially identified as a critical mediator of apoptosis induced by the tumor suppressor p53 and DNA damaging agents (Nakano and Vousden, 2001; Yu et al., 2001). PUMA plays an essential role in p53-dependent and -independent apoptosis in human cancer cells and mice, and mediates apoptosis through the Bcl-2 family proteins Bax/Bak and the mitochondrial pathway (Yu et al., 2003, 2007; Yu and Zhang, 2003). Several studies have convincingly shown that PUMA induction by DNA damage is entirely dependent on an intact p53 gene and is mediated through the two p53-responsive elements in its promoter (Yu et al., 2001; Wang et al., 2007). On the other hand, PUMA is also induced by non-genotoxic stimuli such as kinase activators, endoplasmic reticulum poisons, cytokine withdrawal and growth factor deprivation in several cell types independent of p53 (Yu and Zhang, 2003). Recent studies from us and others have shown that transcription factors such as p73, Sp1 and FoxO3a regulate PUMA induction after serum starvation or cytokine withdrawal in colon cancer cells and lymphocytes, respectively (You et al., 2006; Ming et al., 2008).
In this study, we found that PUMA is induced by three EGFR-targeting agents independent of p53 in HNSCC cells. Further investigation suggested that PUMA functions as a critical mediator of EGFR inhibitor-induced apoptosis in head and neck cancer cells, where p53 family proteins including p73, p63, and the PI3K/AKT pathway serve as key regulators of PUMA induction after EGFR inhibition. Our study provides a molecular mechanism of apoptosis induced by EGFR-targeted therapies in head and neck cancer cells.
PUMA is normally expressed at low basal levels and can be induced by both genotoxic and non-genotoxic stresses (Ming et al., 2008). As >80% of HNSCC overexpress EGFR, we chose to examine PUMA levels after the inhibition of EGFR signaling. HNSCC cell lines were treated with three commonly used EGFR-targeting agents, including the TKIs gefitinib and erlotinib, and the EGFR monoclonal antibody cetuximab. We found that PUMA was induced by gefitinib in the majority (12 out of 13, >90%) of HNSCC lines tested (Figure 1a and Supplementary Table S1), six of which contain mutant p53 (Supplementary Table S1). Erlotinib and cetuximab (C225) also induced PUMA in eight HNSCC cell lines tested (Figure 1b and data not shown). There are at least four species of PUMA transcripts (α, β, γ, and δ) due to alternative splicing. Only the two BH3-encoding isoforms (PUMA-α and PUMA-β) are found to have pro-apoptotic activity, and are detected by this antibody (Nakano and Vousden, 2001; Yu et al., 2001, 2006). Both PUMA-α and PUMA-β were induced after EGFR inhibition in HNSCC cells, whereas the predominant form varied among these lines (Figures 1a and b). Gefitinib-induced PUMA mRNA expression as early as 12 h and the induction peaked at 24 h, which preceded protein induction (Figure 1c).
We then determined whether PUMA induction occurs in vivo using a xenograft model. Established 1483 xenograft tumors were treated with cetuximab (C225) (i.p.), erlotinib (oral gavage), or vehicle (Figure 1d). Both C225 and erlotinib inhibited tumor growth (P = 0.01 and P = 0.08, respectively), with the effects of erlotinib slightly below statistical significance (Figure 1d). PUMA was found to be induced by over 13-fold in the tumors from C225-treated mice and by three-fold in those treated by erlotinib (Figure 1d). The above data indicate that PUMA is induced by EGFR-inhibitors at the transcriptional level in HNSCC cells in vitro and in vivo, irrespective of their p53 status.
We then determined the sensitivity of various HNSCC lines to gefitinib using MTS assay. Five HNSCC cell lines (JHU-012, JHU-019, JHU-022, JHU-029 and 1483) were treated with increasing concentrations (50 nM to 25 μM) of gefitinib for 72 h. Gefitinib achieved a 50% growth inhibition (IC50) at concentrations of <1 μM in only one cell line JHU-029 (Supplementary Table S2 and data not shown). This is consistent with an earlier report that most HNSCC lines are relatively resistant to gefitinib, with IC50s between 10–15 μM (Chun et al., 2006). Interestingly, JHU-029 cells (IC50 at ~0.5 μM) required as little as 0.1 μM gefitinib to induce PUMA, compared with a much higher concentration (around 15 μM) required by JHU-012 cells (IC50 at ~10 μM) (Figure 2a and Supplementary Table S2). PUMA expression was also analyzed in an earlier identified gefitinib-sensitive HNSCC cell line 686LN (P-GC; IC50 at 0.16 μM) and in gefitinib-resistant isogenic sublines R29 and R30 generated by several rounds of selection in vitro (IC50s at ~25 μM) (Figure 2b and Supplementary Table S2) (Muller et al., 2008). Interestingly, PUMA was induced in the parental cells by as little as 0.2 μM gefitinib, but not by up to 25 μM gefitinib in the resistant lines (Figure 2b).
As another BH-3-only protein Bim was recently reported to mediate EGFR-TKI induced apoptosis in lung cancer cells (Costa et al., 2007; Cragg et al., 2007; Gong et al., 2007), we therefore analyzed the expression of Bim and that of three antiapoptotic Bcl-2 family proteins Bcl-2, Bcl-xL and Mcl-1. Bim was induced in JHU-029 and 686LN cells, but not in JHU-012, 686LN R29 or R30 cells (Supplementary Figure S1). The levels of antiapoptotic Bcl-2 family of proteins were not consistently modulated by EGFR-TKI resistance (Supplementary Figure S1).
To determine whether PUMA induction is associated with the degree of inhibition in EGFR signaling, we investigated the effect of gefitinib on ligand-induced EGFR phosphorylation. EGFR phosphorylation was inhibited by gefitinib in a dose-dependent manner in both JHU-012 and JHU-029 cell lines (Figure 2c). EGFR phosphorylation was completely inhibited by 0.2 μM gefitinib in JHU-029, and by ~5 μM in JHU-012 cells (Figure 2c). Furthermore, 11 of 13 HNSCC cell lines, but neither of the two keratinocyte primary cultures showed detectable and relatively high expression of EGFR. The levels of EGFR did not correlate with EGFR-TKI sensitivity in these lines (Supplementary Table S1). These results suggest that PUMA induction, but not EGFR levels, is associated with sensitivity to EGFR-TKI.
Gefitinib-induced dose-dependent caspase activation and apoptosis in JHU-012 and JHU-029 cells (Figure 2a). The doses required to induce appreciable caspase-3 activation or apoptosis are comparable to those required to induce PUMA expression (Figure 2a). In order to determine whether PUMA plays a critical role in EGFR inhibition-induced apoptosis, we attempted PUMA knockdown by siRNA. PUMA knockdown significantly blocked gefitinib-induced apoptosis and caspase-3 activation in both JHU-012 and JHU-029 cells (Figure 2d, P<0.01). In addition, stable PUMA knockdown (KD) JHU-012 cells (two independent clones) that we generated were also resistant to gefitinib-induced apoptosis and caspase-3 activation compared with either the control or parental cells (Supplementary Figure S2A and B). These data suggest that PUMA mediates gefitinib-induced apoptosis in HNSCC cells.
Our earlier data indicated that EGFR-targeting agents activate PUMA transcription independent of p53 status (Figure 1 and Supplementary Table S1). The p53 family member p73 was recently shown to regulate the expression of the BH3-only proteins PUMA and Noxa in HNSCC cells (Rocco et al., 2006). We therefore tested whether p73 mediates PUMA induction after EGFR inhibition. p73 was induced by gefitinib in several HNSCC cell lines, whereas p53 levels remained unchanged (Figure 3a). p73 was also induced by the treatment of erlotinib or cetuximab in both JHU-012 and JHU-029 cells (Figure 3a). Interestingly, p73 induction occurred only in the parental 686LN cells but not in the gefitinib-resistant cell lines (Supplementary Figure S3). This induction did not seem to be associated with an obvious increase in p73 mRNA (Supplementary Figure S4A).
We next determined whether PUMA transcription is directly regulated by p73. As several p73 antibodies failed to precipitate endogenous p73, HA-tagged p73β was first transfected into cells to facilitate its detection. After gefitinib treatment, the recruitment of p73 to the PUMA promoter containing two p53-binding sites was found to significantly increase in a time-dependent manner in JHU-012 and JHU-029 cells. In contrast, the binding of p53 to the same region was unaffected by gefitinib treatment (Figure 3b). Using a series of PUMA deletion reporter constructs (Ming et al., 2008), we found that only the reporters containing the two p53-binding sites, such as Frag A, abc and Frag c, were significantly activated by gefitinib treatment (Figure 3c). Furthermore, knockdown of p73 by small interference RNA (siRNA) impaired gefitinib-induced PUMA expression (Figure 3d). These data suggest that p73 activates PUMA transcription after gefitinib treatment through the p53-binding sites.
The PI3K/AKT pathway promotes cell survival, and is a well-established downstream effector of EGFR signaling (Citri and Yarden, 2006). We examined the effects of gefitinib on the PI3K/AKT signaling in relation to PUMA and p73. Gefitinib treatment resulted in decreased AKT phosphorylation in multiple HNSCC cell lines in which PUMA was induced (Figures 4a and and1,1, and data not shown). Overexpression of AKT suppressed PUMA induction by gefitinib (Figure 4b), whereas overexpression of dominant-negative PI3K (p85) alone induced PUMA expression in the absence of gefitinib treatment in both JHU-012 and JHU-029 cells (Figure 4c). The changes in p73 expression followed similar patterns in these experiments (Figures 4b and c). These results suggest that the PI3K/AKT pathway regulates PUMA levels in HNSCC through p73.
The N-terminal-deleted p63 (ΔNp63) is overexpressed in a significant number of HNSCC and acts as a survival factor (Barbieri and Pietenpol, 2006; Rocco et al., 2006). Interestingly, p63 levels were found to be suppressed by gefitinib or erlotinib in several HNSCC cell lines (Figure 5a). This was not attributed to decreased levels in p63 mRNA (Supplementary Figure S4B). More strikingly, the binding of endogenous p63 to the PUMA promoter was very robust in untreated cells, but was significantly inhibited by gefitinib treatment within 36 h (Figure 5b). Furthermore, overexpression of ΔNp63 inhibited the recruitment of p73 to the PUMA promoter after gefitinib treatment (Figure 5b).
Overexpression of ΔNp63, but not the DNA-binding domain deleted mutant (ΔDBD), blocked PUMA transactivation or protein induction by p73β in p53 knockout HCT116 cells (Figure 5c), which ruled out any involvement of p53 in this regulation. Furthermore, expression of ΔNp63, but not ΔDBD, suppressed PUMA induction after gefitinib treatment (Figure 5d). Taken together, these data suggest that ΔNp63 overexpression inhibits PUMA induction at least in part by blocking p73-mediated transcription, and can lead to resistance to EGFR inhibitor-induced apoptosis in HNSCC cells.
The critical role of PUMA in gefitinib-induced apoptosis suggests that manipulation of PUMA may improve the effectiveness of gefitinib. Using adenoviral expression system Ad-PUMA (Yu et al., 2003), we found that PUMA sensitized gefitinib-resistant HNSCC cell lines to apoptosis (Figure 6a and data not shown, P<0.001). We then tested whether pharmacological agents that mimic the BH3 domain can enhance gefitinib-induced apoptosis. We chose gossypol, a polyphenol derived from cottonseed, as its analogs have shown potent antitumor activities in HNSCC in vitro (Oliver et al., 2004) and in vivo (Wolter et al., 2006), and have entered clinical trials (ClinicalTrials.gov). Gossypol and gefitinib alone did not induce significant levels of apoptosis in HNSCC cells at the concentrations tested (Figure 6b). However, their combination induced significantly higher levels of apoptosis in three HNSCC lines, beyond an additive effect (Figure 6b, P<0.01). Another BH3 mimetic HA14-1 also enhanced gefitinib-induced apoptosis in JHU-022 cells (data not shown). As expected, overexpression of Bcl-2 blocked apoptosis induced by gefitinib in both JHU-012 and JHU-029 cells (Figure 6c, P<0.01). Earlier studies showed that gossypol and its analogs bind to multiple Bcl-2-like proteins to displace BH3 peptides (Kitada et al., 2003; Wang et al., 2006a), and HA14-1 inhibits Bcl-2 (Wang et al., 2000). It is therefore possible that additional BH3-only proteins displaced from Bcl-2-like proteins further potentiate gefitinib-induced apoptosis. Our data suggest that the levels of PUMA perhaps with other BH3-only proteins modulate the sensitivities of HNSCC cells to EGFR-TKI through the mitochondrial pathway.
Our data support a model in which PUMA mediates EGFR inhibitor-induced apoptosis in HNSCC (Figure 6d). On binding to EGFR either at its extra- or intra-cellular domain, EGFR inhibitors block phosphorylation of EGFR and inhibit the PI3K/AKT pathway, which leads to increased expression of p73 and its binding to the PUMA promoter and subsequent transactivation. In some HNSCC cells, gefitinib-induced downregulation of oncogenic ΔNp63 can further enhance p73-mediated PUMA transcription. This model is supported by several lines of evidence: PUMA induction is correlated with EGFR-TKI sensitivity and p73 induction; PUMA knockdown results in resistance to gefitinib-induced apoptosis; the PI3K/AKT pathway suppresses p73 and PUMA induction; and ΔNp63 antagonizes gefitinib- and p73-mediated PUMA induction.
Our findings have several important implications in understanding the therapeutic mechanisms of response to EGFR-targeting agents. EGFR-targeting agents modulate the levels of p63 and p73 in an opposite manner to increase PUMA expression in HNSCC cells (Figure 6d). Our data therefore support a competition model in which p63 antagonizes p73 and gefitinib-mediated PUMA activation by occupying the PUMA promoter containing p53-binding sites (Figure 6d) (Rocco et al., 2006). Suppression of the PI3K/AKT signaling seems to mediate p73 induction by gefitinib. The changes in the levels of p73 or ΔNp63 do not seem to occur through a transcription-dependent manner (Figure 4 and Supplementary Figure S4). Our data is consistent with an earlier report in which gefitinib treatment resulted in decreased ΔNp63 expression in JHU-012 cells (Matheny et al., 2003). Another study from the same group suggested that ΔNp63 is subjected to PI3 K regulation in primary and immortalized keratinocytes (Barbieri et al., 2003). However, we found that ΔNp63α expression was not affected by blocking PI3K/AKT signaling in HNSCC cells, unlike that of p73 (data not shown).
EGFR-targeting agents have been reported to induce apoptosis in different types of cancer cells including HNSCC (Kalyankrishna and Grandis, 2006), though the mechanisms are not well understood. Activation of pro-apoptotic molecules or suppression of the PI3K/AKT (Sordella et al., 2004) pathway have been described (Kalyankrishna and Grandis, 2006). Induction of Bax and activation of caspase-8 were reported in DiFi colon carcinoma cells (Liu et al., 2000). EGFR inhibition led to the activation of BH3-only protein Bad (She et al., 2005) or enhanced the expression of Bim (Costa et al., 2007; Cragg et al., 2007; Gong et al., 2007) in lung cancer cells that contain oncogenic EGFR mutations. Despite the prevalence of EGFR overexpression in HNSCC, mutations in EGFR are extremely rare if present at all. Our data provide direct evidence that PUMA is important in EGFR–TKI-induced apoptosis in HNSCC cells. We also noted that Bim was induced in the two gefitinib-sensitive HNSCC cell lines, but not in the resistant cell lines (Supplementary Figure S1). Taken together, induction or activation of BH3-only proteins by EGFR-TKIs can lead to mitochondria-mediated apoptosis in cancer cells.
The clinical response rates to EGFR-targeted therapies are generally between 10–20% in HNSCC (Bonner et al., 2006; Choong and Cohen, 2006). The discovery of biomarkers that can predict response to therapy would facilitate the identification of HNSCC patients who are likely to benefit from these treatments. Only 2 out of 13 HNSCC cell lines tested showed IC50s <1 μM, resulting in detectable PUMA induction. Much higher concentrations of EGFR-TKI are needed to cause measurable apoptosis or PUMA induction in the resistant cell lines. Studies so far have not indicated EGFR levels as a good correlate with the clinical response to EGFR-TKIs (Supplementary Table S1) (Kalyankrishna and Grandis, 2006). Given the extensive crosstalk among the ERBB family kinases and their overlapping specificity to ligands (Citri and Yarden, 2006), it is perhaps more practical to examine their downstream effectors. The levels or status of the PI3K/AKT pathway, p73, p63, PUMA and other BH3-only proteins are reasonable candidates for future correlative studies using clinical samples.
Defective apoptosis is a hallmark of cancer (Hanahan and Weinberg, 2000). Many genetic and epigenetic changes promote survival of cancer cells and provide ideal targets for developing new anticancer drugs, as such drugs may selectively kill cancer cells while sparing normal cells whose survival does not rely on such changes. Much of the data indicate that the levels of BH3-only proteins are critical determinants of the apoptotic threshold in cancer cells (Yu and Zhang, 2004; Labi et al., 2006). Earlier studies by us and others showed that elevated PUMA expression is toxic to cancer cells and sensitizes them to chemotherapy and radiation (Yu et al., 2006; Wang et al., 2006b; Sun et al., 2007). Reduced PUMA expression was reported to correlate with therapeutic resistance and poor survival in some tumors (Jeffers et al., 2003; Villunger et al., 2003; Yu et al., 2003; Karst et al., 2005). In light of the observation that PUMA, or the BH3 mimetics, sensitizes HNSCC cells to gefitinib-induced apoptosis, the combinations of EGFR-targeted therapies with BH3 mimetics are anticipated with the development of more selective BH3 mimetics such as ABT-737 (Zhang et al., 2007).
The head and neck cancer cell lines (Supplementary Table S1) were obtained from the University of Pittsburgh Cancer Institute (UPCI) Head and Neck Cancer program. None of these lines was derived from EGFR inhibitor-treated patients. Two gefitinib-resistant 686LN cell lines (R29 and R30) have been described (Muller et al., 2008). The p53 knockout HCT 116 colon cancer cells have been described (Bunz et al., 1999). All cell lines were maintained at 37 °C in 5% CO2. Cell culture media included DMEM (Mediatech, Herdon, VA, USA) for the 1483 cells, RPMI 1640 (Cellgro, Herdon, VA, USA) for the JHU cell lines, DMEM/F12 (1:1) for 686LN cell lines, and EMEM (Invitrogen, Carlsbad, CA, USA) for the UPCI: SCC cell lines. The cell culture media were supplemented with 10% FBS (HyClone, Logan, UT, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The EGFR antagonists used included gefitinib (AstraZeneca, Wilmington, DE, USA), erlotinib (Genentech, South San Francisco, CA, USA) and cetuximab (C225) (ImClone, New York, NY, USA). EGF was purchased from R&D Systems (Minneapolis, MN, USA). Gossypol and HA14-1 were from Sigma (St Louis, MO, USA) and Axxora LLC (San Diego, CA, USA), respectively. All drugs were dissolved in DMSO and diluted to the appropriate concentrations with cell culture media before use. For combination treatments with Ad-PUMA, cells were infected with adenoviruses for 24 h followed by drug treatment in virus-free media. All cells were plated in 12-well plates 18 h before treatment unless specified.
Adherent and floating cells were harvested and analyzed for apoptosis by nuclear staining with Hoechst 33258 (Invitrogen) and by flow cytometry (Sun et al., 2007). Cell growth was measured by the MTS assay in 96-well plates (Sun et al., 2007). Each experiment was carried out in triplicate and repeated at least twice.
Total RNA was isolated from HNSCC cells or xenografts using the RNAgents Total RNA Isolation System (Promega, Madison, WI, USA). First-strand cDNA was synthesized from 5 μg of total RNA using Superscript II reverse transcriptase (Invitrogen). The primers used for PUMA, p63, p73 and GAPDH were described in Supplementary Table S2. The conditions for PCR and real-time PCR are available on request (Wu et al., 2007; Qiu et al., 2008).
Immunoblotting was carried out using total cell lysates as described (31). The antibodies used for western blotting included polyclonal antibodies against PUMA (12), p73 (NeoMarkers, Fremont, CA, USA), p53 (DO1), p63, HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Mcl-1, Bcl-xL, total-EGFR (BD Biosciences, San Diego, CA, USA), Bcl-2 (DAKO, Carpinteria, CA, USA), Myc, phospho-AKT (Ser473), total-AKT, phospho-EGFR (Tyr1068), V5 (Invitrogen), α-tubulin, and active caspase-3 (Stressgen Bioreagents, Ann Arbor, MI, USA).
The AKT expression plasmid was purchased from Millipore (Bedford, MA, USA), and the dominant-negative PI3K plasmid (p85) was a gift from Dr Chuanshu Huang (New York University) (Huang et al., 1997). The expression constructs for p63, the DNA-binding domain mutant (ΔDBD), were generated by cloning respective PCR fragments into pcDNA3.1/V5-His vector (Invitrogen), The inserts were verified by DNA sequencing. The primers (Supplementary Table S2) and details for cloning are available on request. PUMA reporters have been described (Ming et al., 2008). The pTAp73β expression construct was from Dr. Carol Prives (Columbia University, New York, NY, USA), and the Bcl-2 expression construct has been described (Pietenpol et al., 1994). Reporter assays were carried out in 12-well plates as described (Yu et al., 2001). The normalized relative luciferase activities (RLA) were plotted. All reporter experiments were carried out in triplicate and repeated three times. The amount of total DNA in transfection is constant in each set of experiments. In some experiments, 0.9 μg of pcDNA-p63 (V5) and/or 0.1 μg pTAp73β were used. Details are described in the Supplementary material.
ChIP was carried out by using the Chromatin Immunoprecipitation Assay kit (Upstate Biotechnology, Lake Placid, NY, USA) according to manufacturer’s instructions with minor modifications (Wang et al., 2007). Antibodies against p63, HA, p53 and isotype-matched IgG (R&D System, Minneapolis, MN, USA) were used for IP. Details and the primers (Supplementary Table S3) are described in the Supplementary material. To analyze the effects of p63 on the recruitment of p73 to the PUMA promoter, JHU-012 cells were transfected with the HA-p73 expression construct alone, or combined with p63 expression construct (1:5 molar ratio) for 18 h, and treated with 15 μM gefitinib for 36 h. The ChIP assay was then carried out.
Cells at 30% confluency were transfected with p73 or PUMA siRNA duplex (Dharmacon, Lafayette, CO, USA) by Lipofectamine 2000 following the manufacturer’s instructions. The target sequences of p73 and PUMA siRNA duplexes were described in Supplementary Table S4. LaminA/C or scrambled siRNA (Dharmacon) was used as a control in these experiments. Twenty-four hours after transfection, the cells were treated with gefitinib for 48 h and harvested for protein or apoptosis analysis.
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. The 1483 cells were implanted into both flanks of 5–6-week-old female athymic nude mice (Harlan, Indiana-polis, IN, USA) as described (Sun et al., 2007), and allowed to establish for 10 days followed by treatment for 2 weeks. Tumor growth was monitored thrice a week using calipers to calculate tumor volumes according to the formula Length × Width2 × 0.52. Animals were killed at the end of the study, and the tumors were harvested and snap frozen for RNA extraction later. Details are described in the Supplementary material.
Statistical analysis was carried out using GraphPad Prism IV software. P-values were calculated by the student’s t-test. P-values <0.05 were considered significant. The means±one standard deviation (s.d.) were displayed in the figures.
We thank other members of our laboratories for helpful discussion and advice. We also thank Drs Cary Wu, Susanne M Gollin (University of Pittsburgh), Bert Vogelstein (HHMI and Sidney Kimmel Cancer Center at Johns Hopkins), David Sidransky (Sidney Kimmel Cancer Center at Johns Hopkins), and Carol Prives (Columbia University) for reagents. This work is supported in part by NIH grant CA129829, P50CA097190 (Head and Neck SPORE career development award) and those from ACGT and FAMRI (J Yu), and by NIH grants CA106348, CA121105, and American Cancer Society grant RSG-07-156-01-CNE (L Zhang).