Necroptosis is a novel mechanism of radiation-induced cell death in anaplastic thyroid and adrenocortical cancers
Matthew A. Nehs, MD,a Chi-Iou Lin, PhD,a David E. Kozono, MD, PhD,b Edward E. Whang, MD,a Nancy L. Cho, MD,a Kaya Zhu, BS,b Jacob Moalem, MD,” Francis D. Moore Jr, MD,ª and Daniel T. Ruan, MD,ª Boston, MA, and Rochester, NY
Background. Necroptosis is a recently described mechanism of programmed cellular death. We hypothesize that necroptosis plays an important role in radiation-induced cell death in endocrine cancers. Methods. Thyroid and adrenocortical carcinoma cell lines were exposed to increasing doses of radiation in the presence of necroptosis inhibitor Nec-1 and/or apoptosis-inhibitor z VAD. H295R cells deficient in receptor interacting protein 1 (RIP1), an essential kinase for necroptosis, were used as controls. Survival curves were generated at increasing doses of radiation.
Results. Nec-1 and zVAD increased cellular survival with increasing doses of radiotherapy in 8505c, TPC-1, and SW13. Both inhibitors used together had an additive effect. At 6 Gy, 8505c, TPC-1, and SW13 cell survival was significantly increased compared to controls by 40%, 33%, and 31 % with Nec- 1 treatment, by 53%, 47%, and 44% with zVAD treatment, and by 80%, 70%, and 65 % with both compounds, respectively (P < . 05). H295R showed no change in survival with Nec-1 treatment. The radiobiologic parameter quasithreshold dose was significantly increased in 8505c, TPC-1, and SW13 cells when both Nec-1 and zVAD were used in combination to inhibit necroptosis and apoptosis together, revealing resistance to standard doses of fractionated therapeutic radiation.
Conclusion. Necroptosis contributes to radiation-induced cell death. Future studies should investigate ways to promote the activation of necroptosis to improve radiosensitivity. (Surgery 2011;150:1032-9.)
From Brigham and Women’s Hospital” and the Dana Farber Cancer Center,b Harvard Medical School, Boston, MA, and the University of Rochester Medical Center,“ Rochester, NY
ANAPLASTIC THYROID CANCER (ATC) and adrenocorti- cal cancer (ACC) are among the most aggressive and lethal human tumors, and effective therapies for these diseases are still lacking. While rare, nearly all patients diagnosed with ATC will die from their disease, and the median survival is ap- proximately 6 months from the time of diagno- sis.1,2 Most patients present with symptoms that may include neck discomfort from a rapidly ex- panding neck mass, vocal cord paralysis, or
Supported by an Osteen Junior Faculty Research Grant (to D.T.R.) from the Department of Surgery at Brigham and Women’s Hospital.
Drs Nehs and Lin contributed equally to this work.
Accepted for publication September 13, 2011.
Reprint requests: Daniel T. Ruan, MD, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, ASBII, Bos- ton, MA 02115. E-mail: druan@partners.org.
0039-6060/$ - see front matter
@ 2011 Mosby, Inc. All rights reserved.
doi:10.1016/j.surg.2011.09.012
dysphagia. These symptoms are the consequences of locally advanced disease that often preclude sur- gical resection. Radioactive iodine is ineffective against ATCs because of the loss of the sodium io- dine symporter through malignant dedifferentia- tion. In addition, ATCs tend to be resistant to chemotherapy and radiotherapy, although some authors have shown limited success in small series with concomitant chemoradiotherapy3 or opera- tions with radiotherapy.
ACC, while also very aggressive, has a somewhat more favorable prognosis, with a 5-year survival rate of approximately 40%.5 Patients with ACC can present with a wide variety of symptoms, including Cushing syndrome, virilization/feminization, signs/ symptoms of excess mineralocorticoids, or abdomi- nal pain from large nonfunctional tumors.6 Surgical resection offers the only chance for cure, but nearly half of completely excised ACCs will recur.7 Adjuvant therapy with the adrenolytic agent mitotane has been used by some authors with varying degrees of suc- cess,8 but no randomized controlled trials have
been performed to show its efficacy. In addition, ad- juvant tumor bed irradiation may decrease local re- currence but has no significant effect on overall survival for ACC.9
Because the prognosis for both ATC and ACC is poor, multimodal therapy including surgery, che- motherapy, and external beam radiotherapy (EBRT) is often used. EBRT uses high-energy photons to induce DNA fragmentation and free radicals in rapidly dividing cells and induce cellu- lar death. While this treatment modality is cur- rently insufficient to cure these diseases, recent investigations have shown that targeted therapy before EBRT can enhance apoptosis, chemosensi- tization, and radiosensitization both in vitro and ex vivo.1º The exact mechanisms of cellular death in response to EBRT, however, have not been fully elucidated. Therefore, a further understanding of the mechanisms of cellular survival and death in response to EBRT for ATC and ACC is needed.
Traditionally, necrosis and apoptosis after mi- totic catastrophe are the principal mechanisms of cellular death from EBRT. Improperly repaired DNA strand breaks cause chromosomal abnormal- ities that interfere with normal segregation during mitosis, which culminates in programmed or spon- taneous cell death.11,12 Necrosis is thought to be a passive process of cellular death that involves mor- phologic changes to cells, including oncosis (in- creased cell volume), chromatin condensation, and swelling of organelles.13 Apoptosis, however, is a coordinated process involving the activation of specific proteases called caspases in response to the binding of specific ligands to so-called “death re- ceptors” on the plasma membrane (eg, Fas). The morphologic changes of apoptosis, in contrast to ne- crosis, include pynknosis (decreased cellular vol- ume), nuclear fragmentation (karyorrhexis), and blebbing of the plasma membrane.13 For years, these 2 processes were thought to represent categor- ically different cellular death mechanisms. How- ever, more recent evidence has revealed coordinated caspase-independent programmed cel- lular death, which has been termed necroptosis. 13-16
Necroptosis is a programmed cellular death pathway that results in the cellular morphology of necrosis but with a signal transduction pathway that more closely resembles that of apoptosis. Fundamental to the process of necroptosis is an effector protein called receptor interacting protein 1 (RIP1), which is a serine/threonine kinase that is known for its role in activation of nuclear factor-KB (NF-KB). While the downstream effectors of this kinase are incompletely understood, RIP1 is a critical protein for the activation of necroptosis.
A
8505-C TPC-1 H295R SW13
+ RIP-1 (74 kD)
+ Actin (43 kD)
PTC specimen #1 (T= 0)
PTC specimen #1 (0 Gy)
B
ATC #1
ATC #2
+ RIP1 (74 kD)
+ Actin (43 kD)
In contrast, RIP1 kinase activity is not necessary for the activation of apoptosis or the NF-KB pathway, making its inhibition useful in specific assessment of the necroptosis pathway. The small molecule necrostatin 1 (Nec-1) is a potent inhibitor of RIP1 kinase activity and therefore is a useful inhibitor of necroptosis in vitro. Similarly, apoptosis can be inhibited using the compound zVAD-fmk, a broad inhibitor of caspase function.
Using specific inhibitors of RIP1 and caspase, we investigated the role of necroptosis and apo- ptosis in thyroid cancer cell lines and ACC cell lines treated with external beam radiation. A further understanding of this mechanism of coor- dinated cellular death may lead to novel agents that activate this pathway to enhance the treatment of advanced endocrine cancers.
METHODS
Materials. Mouse anti-human RIP1 and ubiqui- tin antibodies were purchased from BD Pharmin- gen (Franklin Lakes, NJ). Mouse anti-human actin antibody (pan Ab-5) was purchased from Neo- marker (Fremont, CA). Necrostatin (Nec-1), an inhibitor of necroptosis, and zVAD-fmk, a broad- spectrum caspase inhibitor, were purchased from Calbiochem (La Jolla, CA).
TPC-1 cells
8505-C cells
120
120
100
*
Survival Fraction %
*
Survival Fraction %
100
*
*
*
*
80
*
80
*
60
60
*
*
*
40
40
*
20
20
0
0
0
9
2 Gy
4 Gy
6 Gy
0 Gy
2 Gy
4 Gy
6 Gy
DMSO
DMSO
Nec-1
Nec-1
zVAD
zVAD
Nec-1 + zVAD
Nec-1 + zVAD
SW13 cells
H295R cells
120
120
Survival Fraction %
100
*
Survival Fraction %
100
*
*
*
80
*
*
80
60
*
60
*
*
40
*
40
20
20
0
0
0 Gy
2 Gy
4 Gy
6 Gy
0 Gy
2 Gy
4 Gy
6 Gy
DMSO
· DMSO
Nec-1
O- Nec-1
zVAD
zVAD
Nec-1 + zVAD
Nec-1 + zVAD
Cell culture. ATC cell lines TPC-1 and 8505c were obtained from Dr Sareh Parangi (Massachu- setts General Hospital, Boston, MA). Human adre- nocortical cancer cell lines H295R and SW13 were obtained from Dr Gary D. Hammer (University of Michigan Medical School, Ann Arbor, MI). All cells were cultured as described previously.17,18 The morphology of each cell type used for the experi- ments described was determined based on micro- scopic morphology and by growth curve analysis according to the Cell Line Verification Test Recom- mendations (American Type Culture Collection Technical Bulletin No. 8, 2008). TPC-1, H295R, and SW13 cells were used throughout passages 20-25, and 8505-C cells were used throughout pas- sages 38-44 in this study. Cells were subcultured when 80% confluent.
Human thyroid tumor tissue specimen collec- tions. Human papillary thyroid carcinoma (PTC)
tissues and normal adjacent thyroid tissue were obtained from thyroidectomy specimens resected from patients at Brigham and Women’s Hospital (Boston, MA). The specimens were immediately frozen in liquid nitrogen upon removal, and the samples were stored in liquid nitrogen until the time of protein extraction. Tissue lysates were pre- pared using a radio immunoprecipitation assay buffer containing a protease inhibitor cocktail as described previously.17 The institutional review board of Brigham and Women’s Hospital approved this protocol (no. 1999-P-001331/5).
Radioresistance assay. For radiation experi- ments, cells were trypsinized, replated, and incu- bated for an additional 24 hours under standard conditions. Eight hundred cells were plated in the absence or presence of Nec-1 (100 uM) and/or zVAD (100 µM), and then irradiated with doses ranging from 0 to 6 Gy (Gammacell 40 Exactor;
DMSO
Nec-1
ZVAD
Nec-1 +
ZVAD
T=0
0 Gy
6 Gy
0 Gy
6 Gy
0 Gy
6 Gy
0 Gy
6 Gy
1.00
1.03
0.12
1.07
0.74
1.06
0.69
0.91
0.90
+ p-Histone H3 (17 kD)
+ RIP1 (74 kD)
+ Actin (43 kD)
PTC specimen #1
1.00
1.06
0.03
1.12
0.59
1.08
0.66
0.92
0.89
+ p-Histone H3 (17 kD)
+ RIP1 (74 kD)
+ Actin (43 kD)
PTC specimen #2
1.00
1.02
0.06
1.10
1.16
1.01
0.96
0.98
0.97
+ p-Histone H3 (17 kD)
+ RIP1 (74 kD)
+ Actin (43 kD)
PTC specimen #3
Theratronics, Ottawa, Canada; Cs-1371 Gy/min). Forty-eight hours after irradiation, the media were replaced with fresh media and then subjected to clonogenic survival assay. The mean ± standard deviation of triplicate samples was determined for each treatment. These data were analyzed by plot- ting the log of the surviving fraction (number of colonies) vs radioresistance. The surviving frac- tions were graphed and the width of the shoulder region of the survival curves, a classical indication of the relative repair capacity of the cells, was calculated by graphical analysis as the quasithres- hold dose (Dq).19
Ex vivo PTC tumor specimen culture and live/ dead cell determination assay. A piece of each human tissue specimen was excised using a sterile technique. Vital specimens (0.5 mm3) were cul- tured in Millicell cell culture inserts (Millipore, Bedford, MA) containing 600 uL of Dulbecco’s modified Eagle medium/F12 with 15 mM of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma-Aldrich, Saint Louis, MO) supplemented
with GlutaMAX I (Invitrogen, Carlsbad, CA) in the absence or presence of drug treatment at 37°℃ and 5% CO2. The sample was lysed and then subjected to Western blot analysis.
Statistical analysis. Differences between treat- ment groups were evaluated with a 2-tailed inde- pendent Student t test. Each assay was performed in triplicate.
RESULTS
Thyroid and adrenocortical cancer cells express RIP1. We determined the baseline RIP1 expres- sion in ATC and ACC lines, 8505c, TPC-1, H295R, and SW13 cells by Western blot analysis. We observed that RIP1 is robustly expressed in 8505c, TPC-1, and SW13 cells, while rarely ex- pressed in H295R cells (Fig 1, A). Variable expres- sion of RIP1 in these cell lines is ideal for testing the efficacy of Nec-1 in vitro. We also determined the baseline expression of RIP1 in human ATC and PTC samples and found that it is robustly ex- pressed in these tissues (Fig 1, B).
Nec-1 treatment increases radioresistance in ATC and ACC lines. Because the role of necrop- tosis in the cancer cell response to radiotherapy has not been investigated, we performed radio- resistance experiments with the RIP1 kinase inhib- itor Nec-1 to determine if the inhibition of necroptosis changed radioresistance. Because the kinase activity of RIP1 is not essential for apoptosis or NF-KB activation, Nec-1 specifically inhibits necroptosis.20 As shown in Supplemental Fig 1 (available online at www.surgjournal.com/), there is a dose-dependent effect of Nec-1 with increasing concentrations (from 0-500 uM). To show the ef- fects of Nec-1 on radioresistance, we exposed 4 dif- ferent thyroid and adrenocortical cancer cell lines to increasing doses of ionizing irradiation (0, 2, 4, and 6 Gy) with and without Nec-1 (at a constant concentration of 100 µM).
Nec-1 increased cellular survival for 8505c, TPC- 1, and SW13, but not for H295R. Of note, H295R cells are RIP1-deficient, and therefore showed no change in survival with Nec-1 treatment (Fig 2). At 6 Gy, cell survival was significantly increased com- pared to controls by 40%, 33%, and 31% with Nec-1 treatment in 8505c, TPC-1, and SW13 cells, respectively (P < . 05; Fig 2).
At 6 Gy, treatment with zVAD-fmk, an apoptosis inhibitor, increased cellular survival with increas- ing doses of radiotherapy in 8505c, TPC-1, SW13, and H295R cells by 53%, 47%, 44%, and 51%, respectively. Combined treatment of Nec-1 plus zVAD-fmk increased radioresistance in 8505c, TPC- 1, and SW13 cells by 80%, 70%, and 65%,
0 Gy
6 Gy
DMSO
120
Relative Ki-67 positive cell %
100
80
60
40
Nec-1
20
0
DMSO
Nec-1
ZVAD
Nec-1 + zVAD
ZVAD
0 Gy
6 Gy
Nec-1 + ZVAD
respectively. Our results show that necroptosis and apoptosis inhibition exhibit additive effects on radioresistance.
Nec-1 and zVAD treatment increases radioresist- ance in ATCs maintained in acute culture. Cancer cell lines accumulate genetic changes that are not found in primary tumors, and they fail to account for important tumor-stromal cell interactions that influence radiosensitivity. To more closely recapit- ulate human endocrine cancers, we performed radiosensitivity experiments using human thyroid cancer specimens maintained ex vivo. We tested the effects of Nec-1 and zVAD in 3 human papillary thyroid cancer samples treated with EBRT during acute culture maintenance. As shown in Fig 3, 6 Gy of radiation significantly decreased the expression of p-Histone H3, a common marker of prolifera- tion. The inhibition of apoptosis and necroptosis abrogated the decrease in p-Histone H3 observed in EBRT-treated controls. We confirmed that the proliferation occurred in the PTC cells rather than the surrounding stromal elements by using Ki-67 immunohistochemistry (Fig 4). We found that PTC samples that were treated with Nec- 1 and/or zVAD maintained strong Ki-67 immuno- staining at 6 Gy of radiation, whereas control cells
showed minimal expression of Ki-67. These results suggest that both apoptosis and necroptosis are im- portant mechanisms for radiation-induced cellular death.
Nec-1 treatment increases radiation damage repair capacity. Dq is a parameter of cell survival curves that represents radiation damage repair capacity. Specifically, it reflects the dose of radia- tion that a cell can absorb and effectively repair; when exceeded, this results in exponential cell killing. The Dq value of dimethyl sulfoxide-treated 8505c, TPC-1, SW13, and H295R cells were 3.4, 3.3, 3.5, and 3.4, respectively (Table). The Dq values in 8505c, TPC-1, and SW13 cells were increased to 3.8, 3.5, and 3.8 with Nec-1 treatment, 3.8, 3.7, and 3.9 with zVAD treatment, and 4.1, 4.0, and 4.3 with Nec-1 plus zVAD treatment, respectively. In H295R cells, Nec-1 has no impact on Dq value, while zVAD increased Dq value to 3.8. Necroptosis inhibition by Nec-1 increases survival of irradiated cells in those cell lines that express a robust RIP1 level.
DISCUSSION
While considerable progress has been made in the understanding of the molecular derangements
| Dq | |
|---|---|
| TPC-1 | |
| DMSO | 3.39 |
| Nec-1 | 3.79 |
| zVAD | 3.85 |
| Nec-1 + zVAD | 4.1* |
| 8505c | |
| Control | 3.26 |
| Nec-1 | 3.54 |
| zVAD | 3.68 |
| Nec-1 + zVAD | 4.00* |
| SW13 | |
| Control | 3.48 |
| Nec-1 | 3.77 |
| zVAD | 3.89 |
| Nec-1 + zVAD | 4.25* |
| H295R | |
| Control | 3.39 |
| Nec-1 | 3.43 |
| zVAD | 3.74 |
| Nec-1 + zVAD | 4.00* |
*P < . 05 (Student t test).
ACC, Adrenocortical cancer; ATC, anaplastic thyroid cancer; Dq, quasi- threshold dose; Nec-1, necroptosis inhibitor.
of aggressive endocrine cancers, we still have yet to translate these findings into efficacious treatments for ATC and ACC. With targeted molecular ther- apies still under investigation, EBRT remains an important component in the multimodal therapy for many patients with these cancers. Here we have shown that the co-ordinated caspase-independent cellular death pathway, necroptosis, was involved in radiation-induced cellular death for ATC and ACC cell lines.
The thyroid cancer cell lines TPC-1 and 8505c both had robust RIP1 kinase expression as deter- mined by Western blot analysis and showed in- creased radioresistance with Nec-1 treatment with increasing doses of radiation. In these assays, the significant effects of RIP1 kinase inhibition with Nec-1 were observed with 4 and 6 Gy, but not 2 or 0 Gy. The ACC cell line SW13 had a similar expres- sion of Nec-1 by Western blot analysis and radio- resistance response to Nec1 treatment compared with the thyroid cancer lines. The ACC cell line H295R, however, is known to lack the expression of RIP1 kinase, and was used in this study as a control. As expected, we did not observe a significant difference in radioresistance between Nec-1 treat- ment and controls in H295R cells.
Apoptosis inhibition was achieved using the caspase inhibitor zVAD-fmk, and we found that treatment with this caspase inhibitor caused radio- resistance in all 4 cell lines at both 4 and 6 Gy. Interestingly, when both Nec-1 and zVAD were used in combination, we found that the effect was additive. This suggests that both apoptosis and necroptosis are important for the radiation- induced cellular death for ATC and ACC. Inhibit- ing both mechanisms of programmed cell death provide increased opportunities for cells to repair potentially lethal or sublethal DNA damage.
We tested both Nec-1 and zVAD against 3 independent human papillary thyroid cancer sam- ples taken directly from surgical specimens. As shown in Fig 3, 6 Gy of radiation significantly de- creased the expression of p-Histone H3, a com- mon marker of proliferation. Both Nec-1 and zVAD treatment increased cellular proliferation during EBRT, suggesting that both apoptosis and necroptosis are important mechanisms for radiation-induced cellular death. This finding was confirmed by Ki-67 proliferation index immunohistochemistry.
Necroptosis is an active area of investigation. Both the inhibition and the activation of this cellular death pathway could have therapeutic implications depending on the clinical context. For example, inhibition of necroptosis with Nec- 1 has been used as an anti-inflammatory agent to decrease ischemia-reperfusion injury and improve the functional response to traumatic brain injury in mice.21 In contrast, sensitization of malignant cells (eg, ACC and ATC) with a pronecroptosis agent might augment the antitumor effects of EBRT while minimizing the dose of radiation and collateral damage.22 We anticipate that the thera- peutic potential of such compounds will generate significant research in this area.
Activators of necroptosis may also be important adjuvants to targeted therapies that are currently under development. For example, selective inhib- itors of B-RafV600E (PLX4720 and PLX4032) are undergoing preclinical and clinical validation in ATC and melanoma, respectively. These com- pounds have been shown to activate apoptosis and cause cell cycle arrest in melanoma23; however, anti-B-RafV600E-targeted therapy against thyroid cancer cell lines (TPC-1 and 8505c) showed an in- hibition of growth without evidence of apoptosis activation.24 It is possible that a component of the antitumor effect of this type of targeted ther- apy occurs through a necroptosis-meditated path- way, although this specific question has not yet been investigated.
In conclusion, necroptosis is a caspase- independent mechanism of coordinated cellular death. Here we have shown that necroptosis is an important mechanism in radiation-induced cell death for ATC and ACC cells that express RIP1 kinase, and that this pathway has additive effects with apoptosis on radiation-induced cellular death. A potential therapeutic benefit of this pathway could involve an activator of RIP1 kinase or its downstream effectors to radiosensitize tumor cells before EBRT or as a primary treatment alone. Additional investigation is needed to clarify the role of this pathway in the pathogenesis and treatment of advanced endocrine cancers.
We thank Drs Sareh Parangi and Gary Hammer for providing cell lines used in this study.
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18. Cerquetti L, Bucci B, Marchese R, et al. Mitotane increases the radiotherapy inhibitory effect and induces G2-arrest in combined treatment on both H295R and SW13 adrenocor- tical cell lines. Endocr Relat Cancer 2008;15:623-34.
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DISCUSSION
Dr Michael J. Demeure (Scottsdale, AZ): RIP1 mediates the activity of p53. Did you investigate that further as to whether some of this effect might be related to p53?
Dr Matthew A. Nehs (Boston, MA): We did not test for p53, and we did not specifically ask that question.
Dr Douglas Evans (Milwaukee, WI): Can you explain why you picked 6 Gy and correlate that with the higher doses that are generally used in the head and neck re- gion, especially when treating grossly positive margins, for example? And then maybe you could comment on why you used doxorubicin, as opposed to other cytotoxic agents. Lastly, have you used any of the tyrosine kinase inhibitors, since you comment on that in your abstract?
Dr Matthew A. Nehs (Boston, MA): We used 6 Gy on the recommendations of a radiobiologist (Dr Kozono), who is part of our study. We initially tested several differ- ent doses of Nec-1, from 0 to 6 Gy, and determined a sur- vival curve based on his previous examples. With regard to doxorubicin, our laboratory has been investigating this compound as a sensitizer of cells to its chemothera- peutic effects. So, we had some preliminary data and moved forward with these experiments.
With regard to tyrosine kinase inhibitors, Dr Parangi’s group has shown that anaplastic cell lines can cause re- gression in the face of targeted therapy. And while we did not specifically investigate this, it is currently un- known how these compounds cause tumor regression. So, therefore, we postulate this as a potential mechanism for that regression in targeted therapies, but this re- mains unproven.
Dr Douglas Evans (Milwaukee, WI): It will be interest- ing to see how this area progresses because, as you know, the response to the TKIs oftentimes is short-lived. So, what is it that’s unique about necroptosis that is different from other pathways of cell death? Do you have any com- ment on that?
Dr Matthew A. Nehs (Boston, MA): We don’t yet know the answer to this question. It might be that cells develop resistance and gain escape pathways, much as they do to TKIs, anti-BRAF therapies, or proapoptotic compounds. It may be that they develop pathways around the necroptosis pathway.
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