Enhancement of Cetuximab-Induced Radiosensitization by JAK-1 Inhibition
© Bonner et al. 2015
Received: 31 December 2014
Accepted: 2 October 2015
Published: 12 October 2015
It is known that cetuximab (an epidermal growth factor receptor [EGFr] inhibitor) is a radiosensitizer. Also, cetuximab is known to only partially inhibit the signal transducer and activator of transcription – 3 (STAT-3); a mediator of protection from apoptosis. Studies were performed to determine if the radiosensitizing effects of cetuximab could be enhanced with the addition of an inhibitor of STAT-3.
The interaction of JAK-STAT-3 inhibition ([JAK1i]; Calbiochem, LaJolla, CA) and EGFr inhibition (cetuximab) was assessed with and without radiation. Four human head and neck cell lines were studied: UM-SCC-1 and UM-SCC-5, and two modified UM-SCC-5 lines; a STAT-3 knockdown line (STAT-3-2.4) and control (NEG-4.17). Exposure to either 0.5 μg/ml of cetuximab or 1 μM JAK1i for 8 or 24 h resulted in reduced activated STAT-3 (immunoblot), and the combination treatment showed greater reduction in activated STAT-3 compared to the individual treatments. The use of either post-radiation JAK1i (1 μM for 72 h) or post-radiation cetuximab (0.5 μg/ml) enhanced radiation-induced anti-proliferative and apoptotic effects but the greatest enhancement was seen when cells were exposed to both JAK1i and cetuximab post-radiation. Similar results were seen for radiosensitization as assessed by colony formation. Finally, the combination treatment of JAK1i (1 μM) and cetuximab (0.5 μg/ml), following radiation, resulted in an increase of unrepaired radiation-induced DNA double strand breaks at 6 and 24 h after radiation compared to the use of post-radiation JAK1i or cetuximab alone as delineated by neutral comet assay.
These findings suggest that dual inhibition of EGFr (cetuximab) and JAK-STAT-3 (JAK1i) leads to greater radiosensitization than with either cetuximab or JAK1i alone and suggests that this combination treatment may be clinically relevant even for tumors with a marked range of STAT-3 activity.
Cetuximab is an inhibitor of the Epidermal Growth Factor Receptor (EGFr) that binds to the EGFr ligand binding domain, thereby inhibiting downstream EGFr signaling involved in cellular growth . In the clinic, cetuximab has shown modest activity as a single agent for metastatic head and neck cancer (13 % response rate when used alone for recurrent disease) and radiosensitizing activity for locoregionally advanced head and neck cancer [2–4]. Since the EGFr signaling pathway involves multiple downstream phosphorylation reactions and crosstalk with other signaling pathways, it is possible that the anti-tumor effects of EGFr inhibition can be enhanced by inhibiting other downstream effectors of EGFr signaling.
The signal transducer and activator of transcription-3 (STAT-3) is a protein that lies downstream of EGFr and activation of EGFr leads to activated STAT-3, which in turn protects cells from apoptosis. However, it is also known that several other signaling events activate STAT-3. The Janus Kinases (JAK1 and JAK2) are important activators of STAT-3. Furthermore, other signaling cascades, such as the SRC pathway, can activate the JAK/STAT-3 cascade . We have previously shown that cetuximab-induced inhibition of EGFr leads to inhibition of activated STAT-3, but this inhibition is incomplete . It is likely that other activators of STAT-3, such as the Janus Kinases, circumvent more complete STAT-3 inhibition when cetuximab is used alone. Therefore, it is believed that STAT-3 continues to affect downstream protection from apoptosis, and other STAT-3 mediated events such as angiogenesis, even when it is partially inhibited by cetuximab.
In an effort to achieve more complete inhibition of EGFr signaling, we explored the combined inhibition of EGFr and JAK–STAT-3 (dual inhibition) with and without radiation in human head and neck squamous cell cancer cell lines with a variety of STAT-3 expression. One of the tested cell lines had partial knockdown of STAT-3 as previously described [6, 7]. It was determined that JAK-STAT-3 inhibition accentuated the radiosensitizing properties of cetuximab in all cell lines. Although we initially set out to determine whether JAK1i increased the known cetuximab-induced radiosensitizing properties, we found that both agents were radiosensitizers and the radiosensitizing effects were greatest when the agents were given together.
Human head and squamous cell cancer cell lines were grown in Dulbecco’s Modified Eagle’s Medium containing 10 % heat-inactive fetal bovine serum supplemented with 2 μM L-glutamine and incubated in a humidified chamber at 37 °C with 5 % CO2 as previously described [6, 7]. UM-SCC-1 and UM-SCC-5 were obtained from Dr. Thomas Carey at the University of Michigan. UM-SCC-5 cells were used to create stable transfectants of a short hairpin RNA against STAT-3 (STAT-3-2.4 cells). These cells were created by transfecting UM-SCC-5 cells with a pBABE-U6 vector containing STAT-3 short hairpin RNA (shRNA) as previously described . Following transfection, the STAT-3-2.4 cells showed approximately 50 % STAT-3 knockdown as previously described [6, 7]. Control cells were also previously created by transfection with a mutated or negative STAT-3 shRNA form of the short hairpin RNA against STAT-3 (NEG 4.17 cells) and do not show significant STAT-3 knockdown .
Immunoblots were used to analyze the protein expression levels of STAT3 as previously described [6, 7]. UM-SCC-1, −5 and transfected STAT3-2.4 and NEG4.17 cells were assessed for STAT-3, phosphorylated STAT-3 and GAPDH. Cell lysates were prepared and equal amounts of protein were loaded in each gel lane. Separation was performed by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P membrane (Millipore Corp, Bedford, MA). The immunoblots were blocked in 10 % milk–Tris-buffered saline with Tween-20 (TBS-T) (20 mmol/L Tris HCL [pH 7.5], NaCl 137 mmol/L, and 0.05 % Tween-20) for 1 h at room temperature. The primary antibodies of anti-STAT-3, anti-p-STAT-3 (Cell Signaling Technologies, Beverly, MA), anti-EGFr (Sigma-Aldrich, St. Louis, MO), anti-p-EGFr (cell signaling technologies, Beverly, MA) and anti-GAPDH (Santa Cruz Technologies, Inc., Santa Cruz, CA) were incubated overnight at 4 ° C with 2 % milk–TBS-T. The secondary antibody, anti-mouse–IgG–horseradish peroxidase antibody (Sigma Chemical Company, St. Louis, MO) was incubated at room temperature for 1 h. The blots were developed by chemiluminescence (Amersham Life Sciences, Inc., Arlington Heights, IL).
The effects of various treatments on cell proliferation were assessed by a cell proliferation assay as previously described [6, 7]. Briefly, UM-SCC-1, −5, and the transfected STAT3-2.4 and NEG 4.17 cells were plated in a manner to allow multiple days of proliferation. Following the entry of cells into an exponential growth phase, they were exposed to various combinations of cetuximab (0.5 μg/ml), 1 μM Janus Kinase inhibitor ([JAK1i] Calbiochem, LaJolla, CA) and/or radiation (2 Gy). The cells were assessed for cell counts every 24 h. The cells were removed from plates with trypsin and counted using a cell counter (Beckman Coulter, Fullerton, CA). Illustrative experiments demonstrate results following 72 h of growth.
Apoptotic cell death following exposure to various treatments was quantified by using the Annexin V-FITC apoptosis detection kit (BioVision Research Products, Mountain View, CA) as previously described . UM-SCC-1, −5, and the transfected STAT3-2.4 and NEG 4.17 cells were treated with various combinations of cetuximab (0.5 μg/ml), JAK1i (1 μM) and/or radiation (2 Gy) in a manner identical to the cell proliferation assay. Following 72 h of incubation, the cells were isolated and the data were collected using a Becton Dickinson FACScan system. This information was analyzed using CellQuest v3.1 software (Becton Dickinson, San Jose, CA).
Cell survival was assessed by colony formation as previously described [6, 7]. Briefly, cells were plated the day before the start of the experiment in order to allow time for attachment of the cells. The cells were exposed to various doses of radiation, either alone or with exposure to JAK1i, cetuximab or both agents just prior to radiation and during the post-radiation period. The cells were allowed to form colonies over 12–14 days. They were subsequently fixed, stained and counted (colonies of > 50 cells) for colony formation as previously described.
Neutral comet assay
The accumulation of DNA double strand breaks (dsbs) was assessed by the neutral comet assay as previous described . Briefly, exponentially growing UM-SCC-1, −5, and the transfected UM-STAT3-2.4 and NEG 4.17 cells were plated at a concentration of 150,000 cells in 60 mm2 tissue culture dishes. After a 16 h incubation in the presence of cetuximab (5 μg/ml), JAK1i (1 μM) or the combined treatment with or without radiation, the cells were scraped and processed for Neutral Comet Assay at various times following radiation as described by Trevigen (Gaithersberg, MD). Following the staining with SYBR Green I, the slides were viewed by fluorescence microscopy (Evos f1, Advanced Microscopy Group, Bothell, WA). Tail moment and percent DNA in the tail were analyzed using CometScore Freeware.
All experiments were performed with at least three independent experiments. Representative immunoblots were selected for presentation. For experiments other than the immunoblots, results were expressed as the mean ± standard error (SE). For the cell proliferation and apoptosis assays, the statistical significance between treatment groups was determined using a two-way ANOVA assessment with software from GraphPad Prism, San Diego, California. Overall comparisons of certain conditions were made using comparisons of all four cell lines in a simultaneous two-way ANOVA assessment.
This research did not involve human subject.
Since STAT-3 is known to protect cells from apoptosis, the above noted treatment-related effects on cell proliferation were further assessed to determine if they correlated with apoptotic events. Cetuximab and/or JAK1i were tested with and without radiation (Fig. 5). The results correlated with the cell proliferation findings. As with the cell proliferation studies, greater apoptosis was seen when JAK1i was added to the combination of cetuximab and radiation. The overall levels of significance for the comparisons of cetuximab/JAK1i with RT vs. either cetuximab with RT [p = 0.0001], or JAK1i with RT [p = 0.0001], were pronounced. These effects were statistically significant for all individual cell lines except the comparison of cetuximab/JAK1i with RT vs. cetuximab and RT in the control transfectants (NEG 4.17 cells). As with the proliferation studies, there was a suggestion that cetuximab/JAK1i and RT produced more apoptosis than cetuximab/JAK1i, but it was not significant [p > 0.05]. Once again, this comparison was significant when cells were exposed to lower concentrations of JAK1i (0.5 μM) as shown in Fig. 4. In summary, these findings correlated with the cell proliferation studies and demonstrated increased enhancement of radiation-induced apoptosis with the combination of cetuximab/JAK1i compared to the use of either agent alone.
Human head and neck squamous cell carcinoma cancer cells show high levels of EGFr expression and it is believed that this expression plays an important role in the EGFr-mediated receptor tyrosine kinase signaling that controls cellular proliferation and can lead to the aggressive growth of these tumors . Increased expression of EGFr has correlated with a poor prognosis for patients with head and neck cancer treated with conventional therapies . The significance of EGFr in the growth of head and neck malignancies led many investigators to explore the use of anti-EGFr treatments in this disease. These efforts proved fruitful as anti-EGFr treatments have been effective as single agent therapies or in combination with chemotherapy for patients with metastatic disease [2, 4]. Additionally, the inhibition of EGFr has resulted in clinically significant radiosensitization for locoregionally advanced tumors .
Even though anti-EGFr treatments, such as cetuximab are effective in head and neck cancer, it is known that the EGFr pathway receives additional signaling input from other parallel growth pathways . It is believed that these parallel growth pathways can partially circumvent the anti-proliferative effects of anti-EGFr agents, such as cetuximab, by activating proteins that are downstream of EGFr. Therefore, we sought to enhance the anti-proliferative and radiosensitizing effects of cetuximab by adding an inhibitor of STAT-3 which is an important downstream tyrosine kinase that facilitates protection from apoptosis. Others have targeted STAT-3 in human tumor models using various techniques. Grandis, et al., stably transfected human head and neck cancer cells with dominant negative mutant STAT-3 constructs. This alteration resulted in these constructs failing to proliferate . In contrast, a similar dominant negative construct for STAT-1 did not alter the proliferation of the cells.
Recently, this same group, Sen et al. , further assessed the effect of targeting STAT-3, utilizing decoy nucleotide sequences that interfere with STAT-3 mediated DNA binding, on the growth rate of cetuximab-sensitive parental T24 squamous cell cancer cells and T24 cells with acquired resistance to cetuximab. The targeting of STAT-3 decreased proliferation in both the cetuximab sensitive and cetuximab resistant cells. These cells were then grown as xenografts and the combination of cetuximab and STAT-3 decoy was assessed. Treatment of either cetuximab-sensitive or cetuximab-resistant cells with cetuximab and STAT-3 decoy resulted in significant reductions in tumor volume compared to cetuximab with a control mutant decoy. They also showed that STAT-3 levels were increased in squamous cell tumors (patient samples) that recurred following cetuximab treatment. Due to the above findings, they hypothesized that the application of STAT-3 inhibitors may be useful in circumventing cetuximab resistance. They did not examine the use of radiation or cetuximab-induced radiosensitization in these experiments.
The work of Sen et al., may serve as appropriate preliminary preclinical data to consider combining inhibitors of EGFr and STAT-3 for incurable tumors . However, adding JAK1i to the combination of cetuximab and radiation could potentially enhance the cure rate of cetuximab and radiation for patients with locoregionally advanced disease. In fact, cetuximab is a known radiosensitizer and only partially inhibits the activation of STAT-3 and hence, we employed a combination of cetuximab and a JAK1-STAT-3 inhibitor (JAK1i) with or without radiation. This approach showed that the two agents interacted to enhance the inhibition of cellular proliferation and promote apoptosis compared to either agent alone. These results corroborated the results of Sen et al. . Furthermore, the addition of JAK1i enhanced the radiosensitizing effects of cetuximab in human head and neck cancer cells. Finally, our results showed that the combination of the two agents inhibited radiation-induced DNA repair to a greater extent than either agent alone. These effects were demonstrated in four human head and neck squamous cell cancer cell lines including STAT-3-2.4 cells which showed 50 % STAT-3 knockdown as a result of stable transfection of a short hairpin RNA against STAT-3. This latter result increases the potential ramifications of this study. It is very possible that this dual approach to the inhibition of EGFr signaling (that includes anti-STAT-3) may be clinically relevant for tumors with low to high levels of STAT-3 activation.
The findings that the addition of JAK1i to cetuximab and radiation enhanced the anti-proliferative, apoptotic and radiosensitizing effects of radiation and the accompanying result that there was no appreciable difference in this effect for STAT-3 knockdown cells vs. parental cells, may not have been expected. One might have expected JAK1i to be less of a radiosensitizer or add less to the cetuximab-radiation interaction in the STAT-3 knockdown cells. However, this was not the case. These results suggest that even though STAT-3 expression may be reduced in a particular cell line, STAT-3 remains a significant partner with EGFr (and other receptors) regarding the sensitivity of the cell to radiation. It will also be important to investigate whether STAT-3 is a more important driver of EGFr signaling in cell lines with certain molecular characteristics. This information could help investigators exploit STAT-3 inhibition as a clinical target for personalized medicine approaches. Our study did not provide information in this regard. The STAT-3 knockdown cells and parental cells showed similar effects for the studied conditions.
The aforementioned studies were performed based on previous work that demonstrated cetuximab radiosensitized human head and neck squamous cell cancers . The previous findings that cetuximab was a radiosensitizer for squamous cell carcinomas initiated many additional laboratory and clinical investigations in the early 2000’s. Our group reported that cetuximab enhanced radiation-induced apoptosis in human squamous cell cancer, but we did not explore the induction or repair of radiation-induced DNA damage in those early studies. However, following the demonstration that cetuximab was clinically useful as a radiosensitizer , others reported that cetuximab inhibited radiation-induced DNA dsb repair . Although cetuximab alters many signaling pathways that could affect radiosensitivity, the potential role of DNA repair in cetuximab-induced radiosensitization for certain cell populations led to the DNA repair studies reported herein. The findings that the addition of JAK1i to cetuximab and radiation resulted in greater radiosensitization and greater slowing of the repair of radiation-induced DNA dsbs are intriguing. Cetuximab’s known effect of slowing the repair of radiation-induced DNA dsbs is hypothesized to be involved in at least a portion of cetuximab-induced radiosensitization in many cell lines. Cetuximab also influences cellular processes such as cell cycle, apoptosis, and angiogenesis. It is likely that these effects play a role in cetuximab-induced radiosensitization as well [16, 17]. As with cetuximab, it is entirely possible that JAK1i creates many effects (due to altered signaling of several pathways) that are involved in radiosensitivity, but our results suggest that greater slowing of the repair of radiation-induced DNA dsbs contributes to the JAK1i - mediated enhancement of cetuximab-induced radiosensitization.
Others have explored the dual inhibition of molecular targets as a method of enhancing the effects of radiation in head and neck cancer by targeting various aspects of EGFr signaling. It is known that EGFr signaling promotes angiogenesis . Cetuximab leads to a partial decrease in angiogenesis for human head and neck cancer . (This partial cetuximab-induced inhibition is similar to cetuximab’s effect on STAT-3). It is believed that cetuximab-mediated inhibition of EGFr leads to partial reduction in angiogenesis through downregulation of HIF-1a and Notch 1 . Since preclinical studies have shown that cetuximab partially inhibits angiogenesis and this inhibition appears to hinder tumor growth, investigators have been interested in combining cetuximab with anti-VEGF agents, such as, bevacizumab. Additionally, there has been interest in combining both agents with radiation in head and neck cancer. Recently, the Memorial Sloan Kettering group treated 30 patients with locoregionally advanced head and neck cancer with cetuximab (400 mg/m2 loading dose, followed by 250 mg/ml weekly), bevacizumab (15 mg/kg, day 1 and 22) and cisplatin (50 mg/m2, day 1, 2, 22 and 23) concomitantly with radiotherapy (70 Gy). The early results have been promising with an overall survival of 92.8 % at 2 years . Further work will be necessary to determine if the use of this particular combination of dual inhibitors of molecular targets, for the purpose of enhancing radiosensitization, is an advance in the clinical arena.
Additional investigations have employed dual radiosensitizers in head and neck cancer by combining cisplatin with targeted agents. (It is noteworthy that reduced cisplatin doses were employed in the above noted clinical trial with cetuximab, bevacizumab and radiation). Cisplatin has long been used as a radiosensitizer in head and neck cancers. After cetuximab was found to be a radiosensitizer for head and neck cancer, interest arose in combining cisplatin and cetuximab as dual radiosensitizers. The Radiation Therapy Oncology Group (RTOG) recently reported the results of a large phase III study that explored the use of cisplatin-radiotherapy with and without cetuximab for patients with locoregionally advanced head and neck cancer. Unfortunately, the addition of cetuximab to the combination of cisplatin and radiotherapy did not result in a benefit for patients . In the future, combinations of targeted agents with radiotherapy could prove to be an area of impactful investigation as there may be complementary targeted agents that enhance tumor kill without significantly increasing toxicity.
Our work and the work of others suggest that combinations of agents that target overlapping signal transduction pathways may be a means to address relative resistance to single agents . The present study suggests that the combination of inhibitors of EGFr and STAT-3 enhances the effects of radiation in head and neck cancer compared to the use of either agent alone. Inhibitors of STAT-3 are being investigated  and these agents could be combined with cetuximab in future clinical trials.
Funding for this project was provided by the Department of Radiation Oncology at The University of Alabama at Birmingham, Birmingham, Alabama.
The authors wish to acknowledge Gwen Sims for preparation of the manuscript.
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