Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

doi:10.1016/j.ijrobp.2008.08.043

International Journal of Radiation OncologyBiologyPhysics

Volume 73, Issue 1, 1 January 2009, Pages 288-295

https://doi.org/10.1016/j.ijrobp.2008.08.043 Get rights and content

Purpose

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC.

Methods and Materials

The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays.

Results

STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 × 10⁻⁸ for clear cell; and p = 3.6 × 10⁻⁴ for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines.

Conclusion

This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Introduction

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy. Conventional radiotherapy (1.8–2.0 Gy/d) was thought to have little use in the management of primary kidney cancer in terms of cure. In patients with metastatic disease, conventional radiotherapy is effective in palliation of symptoms, but it results in a subjective or objective response in only 50% of patients. Thus, it is imperative to understand the molecular mechanism underlying this radioresistance. Basic mechanisms of signal transduction and DNA repair after irradiation have been studied in simple genetic model systems and in cells derived from radiosensitive patients with genetic disorders such as ataxia telangiectasia 1, 2. Although these investigations have elucidated important basic principles and pathways of the response of eukaryotic cells to radiation damage, a significant effect on clinical management requires more preclinical application or translational studies. Continuing exploration of radioresistance targets in tumors and their manipulation to improve radiotherapy is thus needed.

Renal cell carcinoma is a histologically heterogeneous disease. Several subtypes of RCC have been described, including clear cell, papillary, chromophobe, collecting duct, and unclassified, which did not fit with any other diagnosis. Clear cell RCC is the most common subtype (75–80%), followed by papillary RCC (10–15%) and chromophobe (4–6%). In addition, rarer types of RCC have been morphologically defined, including renal medullary RCC, Xp11 translocation carcinoma, carcinoma associated with neuroblastoma, mucinous tubular carcinoma, and spindle cell carcinoma. Each subtype appears to have a distinct molecular signature reflecting their differences, not only in morphology, but also in biology and clinical behavior (3).

Recently, signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, has been implicated in the presence of radioresistance, first demonstrated in a head-and-neck cancer cell line (4). To date, its involvement in RCC in general and its specific relationship with radioresistance in RCC have never been explored. Using our extensive microarray gene expression data (5), we examined the level of STAT1 expression in the two most common subtypes of RCC (i.e., clear cell and papillary). Immunohistochemistry of STAT1 and phosphorylated STAT1 were performed on RCC tissue arrays to validate the microarray data. We then studied the expression level of STAT1 and the cellular effects and response to radiation on two representative cell lines of each subtype after STAT1 inhibition, both by RNAi and fludarabine, a nucleoside analog used as a chemotherapeutic agent that can reduce the level of STAT1 protein. Finally, we examined the expression of signal transducer and activator of transcription 3 (STAT3) in both human RCC and the cell lines to establish whether the known relationship between STAT1 and STAT3 exists in human RCC and whether the radioresistant effect is related to either STAT1 or STAT3 or a combination.

Section snippets

Examination of STAT1 expression in RCC vs. normal kidney using microarray gene expression database

We compared the expression of STAT1 and STAT3 in clear cell and papillary RCC and normal kidney tissue samples using our extensive gene expression profiling database of renal tumors (5). For this study, 164 clear cell and 47 papillary RCC samples and 15 normal kidney samples were included.

Immunohistochemistry

Paraffin tissue blocks from 34 clear cell RCC samples (30 conventional and 4 with sarcomatoid features) and 12 normal adjacent kidney tissue samples were studied, and tissue microarray analysis was performed.

STAT1 differentially overexpressed in human RCC by microarray analysis

The STAT1 expression data in human clear cell and papillary RCC and in corresponding normal renal tissues is shown in Fig. 1A. The mean and standard errors for STAT1 gene expression were calculated. A two-tailed Student's t test was calculated, and the difference was determined to be statistically significant at the 95% confidence interval (p = 1.5 × 10⁻⁸ for clear cell and p = 3.6 × 10⁻⁴ for papillary). This is the first study to report the overexpression of STAT1 in human RCC. No elevation

Discussion

Renal cell carcinoma is traditionally considered to be radioresistant. The failure in treating tumors by radiotherapy has been ascribed to intrinsic tumor cell radioresistance and tumor microenvironmental factors, such as hypoxia, which favors tumor cell survival after irradiation 8, 9. An understanding of the molecular mechanisms underlying tumorigenesis and the cancer cell response to external factors, including irradiation, is necessary to develop more rational and effective treatment. A

Acknowledgments

We thank the Cooperative Human Tissue Network of the National Cancer Institute for providing the renal tumor cases. We also thank David Nadziejka for proofreading and Sabrina Noyes for manuscript preparation and submission.

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Metastatic renal cancer has a poor prognosis because of the limited impact of usual treatment modalities, and notably radiotherapy. Renal cell carcinoma is traditionally considered to be radioresistant, and conventional radiotherapy fraction sizes of 1.8 to 2 Gy are thought to have little role in its management. Technological advances in radiation oncology have led to stereotactic approaches that overcome radio resistance mechanisms of renal cancer cells and could be successful. The technical ability of applying high dose per fraction, leads to a distinct biological response which is different from the one observed with conventional irradiation through high responses rates. The increased radiobiological effect is attributed to endothelial apoptosis triggered by high fractional dose. The combination of such radiotherapy regimens with targeted drugs paves the way for new therapeutic opportunities.
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In order to improve the therapeutic efficiency and reduce the damage to adjacent normal tissues in cancer treatment, the radiotherapy are always combined with agents to increase cytotoxicity or radiosensitivity. Since the therapeutic effect of radiation for patients with RCC is rarely satisfactory, it is imperative to discover novel and potential radiosensitizer to increase the efficacy of palliative radiotherapy [17]. Curcumin has been reported to be involved in the radiosensitivity enhancement in several cancer cells such as colorectal cancer, prostate cancer, and hepatocellular carcinoma [18,19].
The radiation resistance of renal cell carcinoma (RCC) remains the primary obstacle to improve patient survival. This study aimed to investigate the effects of curcumin on the radiosensitivity of RCC cells. Human RCC cell (ACHN) was exposed to irradiation (IR) and/or curcumin treatment. Cell viability, DNA repair, cell cycle, and apoptosis, were evaluated by MTT, immunofluoresence staining and flow cytometry. Moreover, ACHN cells were xenografted into nude mice and subjected to IR and/or curcumin treatment. The expression of NF-κB signaling related proteins in ACHN cells and xenografts was detected by western blot analysis. The results showed that curcumin significantly increased radiosensitivity of ACHN cells by inhibiting the cell proliferation and DNA damage repair, causing cell cycle arrest at G2/M phase, inducing apoptosis in vitro, and suppressing the growth of xenografts in vivo. In addition, curcumin enhanced radiosensitivity was through markedly inhibiting IR-induced NF-κB signaling by modulating the related protein expressions including NF-κBP65, I-κB, VEGF, COX2, and Bcl-2 in ACHN cells, which was further strengthened by NF-κB inhibitor PDTC treatment. Thus, curcumin may confer radiosensitivity on RCC via inhibition of NF-κB activation and its downstream regulars, suggesting the potential application of curcumin as an adjuvant in radiotherapy of RCC.
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While in some instances STAT1 can increase apoptosis through the activation of pro-apoptotic genes and suppression of anti-apoptotic proteins produced by the NF-κB pathway, its constitutive overexpression has also been shown to promote cell survival likely via the modulation of pathways activated by ATM, including Chk2 and NBS1 [148–150]. This overexpression of STAT1, which is often seen in cancers, is likely linked to their observed radioresistance as knockdown of STAT1 has been shown to increase radiosensitivity in the cells of squamous cell carcinoma [148] and in renal cell carcinoma [145]. Researchers have suggested that this radiosensitization is a result of the modulation of IL-6 and IL-8 as the expression of these cytokines greatly diminishes following the downregulation of STAT1 [144].
Despite recent advances in radiotherapy, a majority of patients diagnosed with pancreatic cancer (PC) do not achieve objective responses due to the existence of intrinsic and acquired radioresistance. Identification of molecular mechanisms that compromise the efficacy of radiation therapy and targeting these pathways is paramount for improving radiation response in PC patients. In this review, we have summarized molecular mechanisms associated with the radio-resistant phenotype of PC. Briefly, we discuss the reversible and irreversible biological consequences of radiotherapy, such as DNA damage and DNA repair, mechanisms of cancer cell survival and radiation-induced apoptosis following radiotherapy. We further describe various small molecule inhibitors and molecular targeting agents currently being tested in preclinical and clinical studies as potential radiosensitizers for PC. Notably, we draw attention towards the confounding effects of cancer stem cells, immune system, and the tumor microenvironment in the context of PC radioresistance and radiosensitization. Finally, we discuss the need for examining selective radioprotectors in light of the emerging evidence on radiation toxicity to non-target tissue associated with PC radiotherapy.
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Despite the simplistic view of STAT1 as an immunogenic signalisation pathway in antagonistic relationship with immunosuppressive STAT3, its role in the modulation of antitumor immune response is quite ambiguous: STAT1 activation in tumor-associated macrophages lead to T-lymphocytes apoptosis and restrain specific immune activation (Kusmartsev and Gabrilovich, 2005). Moreover, STAT1 expression is regarded as an intrinsic factor of radioresistance in renal cancer cells that can be inhibited both by zoledronic acid (Kijima et al., 2013) and fludarabine (Hui et al., 2009). STAT3, that is constitutively activated in 70% of solid tumors (Pensa et al., 2008) abort immune response mainly by inhibiting the maturation of antigen presenting cells (APCs), accounting for impaired DC-mediated induction of T cell responses (Yu et al., 2007), recruitment of MDSCs and Tumor-associated Macrophages (TAM2) with acquisition of a M2 phenotype (see dedicated paragraph below).
Major advances in the knowledge of cancer biology and its interactions with tumor immune environment led to the emergence, in the last five years of new immunotherapy-based treatment strategies in cancer patients. At the same time, improvement in radiation technique and progress in radiobiology allowed in the last decade to expand the applications of radiotherapy in a growing number of settings. At present, there are strong theoretical basis to propose immune-enhanced radiation therapy that may represent in the future a new paradigm of treatment, combining the intrinsic power of radiotherapy to elicit a specific, systemic, tumor-directed immune response with modern highly conformal and precise dose delivery, in order to maximize response at the major site of disease and obtain durable disease control. The aim of this review is to describe the principal mechanisms of immune modulation of response to radiation and investigational strategies to harness the potential of radiation-inducible immune response: radiation therapy is expected to be not just a local treatment but the cornerstone of a multimodal strategy that might achieve long-lasting tumor remission at the primary site and systemic efficacy metastatic lesions.
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Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways.
The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed. Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay.
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: Supported by a research grant from the Methodist Hospital Research Institute, and also the Gerber Foundation, Hauenstein Foundation, Michigan Economic Development Corporation, and Michigan Technology Tri-Corridor to B. T. Teh. Supported by National Natural Science Foundation of China for Young Scholar (30800280) to Z. Hui.

: Presented in part at the 48th Annual Meeting of the American Society for Therapeutic Radiology and Oncology, November 5–9, 2006, Philadelphia, PA.

: Conflict of interest: none, except B. S. Teh received a Methodist Hospital Research Institute research grant.

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Biology ContributionRadiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

Purpose

Methods and Materials

Results

Conclusion

Introduction

Section snippets

Examination of STAT1 expression in RCC vs. normal kidney using microarray gene expression database

Immunohistochemistry

STAT1 differentially overexpressed in human RCC by microarray analysis

Discussion

Acknowledgments

J Urol

Cancer Lett

J Biol Chem

Int J Radiat Oncol Biol Phys

Cell Signal

Cell-cycle checkpoints and cancer

Nature

Regulation and mechanisms of mammalian double-strand break repair

Oncogene

Molecular subclassification of kidney tumors and the discovery of new diagnostic markers

Oncogene

STAT1 is overexpressed in tumors selected for radioresistance and confer protection from radiation in transduced sensitive cells

PNAS

STAT1 activation in squamous cell cancer of the oral cavity: A potential predictive marker of response to adjuvant chemotherapy

Cancer Lett

Hypoxia—A key regulatory factor in tumour growth

Nat Rev Cancer

Radiobiology for the radiobiologist

Functional genomics as a window on radiation stress signaling

Oncogene

The evolution of diverse biological responses to DNA damage: Insights from yeast and p53

Nat Cell Biol

Antiangiogenics: The potential role of integrating this novel treatment modality with chemoradiation for solid cancers

J Clin Oncol

Biology Contribution
Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma