Genes associated to radiation response in NSCLC from genomics data
Lung cancer remains as one of the most aggressive cancer types with nearly 1.6 million new cases worldwide each year. There are an estimated 222,520 new cases and 157,300 deaths from lung cancer in the United States in 2010 . Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, comprising three major histological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Chronic exposure to carcinogens drives genetic and epigenetic damage that can result in lung epithelial cells progressively acquiring growth and/or survival advantages, giving as a result the generation of tumor cells. Studies have shown that some specific molecules contribute to sporadic tumors of lung cancer; even now, they are useful as predictive biomarkers. Mutations in at least one of the established lung cancer driver genes including
2. Lung cancer
Lung cancer remains as one of the most aggressive cancer types with nearly 1.6 million new cases worldwide each year. In 2010, in the United States were estimated 222,520 new cases and 157,300 deaths from lung cancer . Non-small cell lung cancer (NSCLC) subtype represents 85% of all cases of lung cancer, while small cell lung cancer (SCLC) subtype comprises 15%. Histologically, NSCLC is classified as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. This classification has important implications for the clinical management and prognosis of the disease . Yet early detection methods are not extensively used in the wider population, malignancy is most commonly diagnosed at a late stage resulting in poor patient survival. Overall 5-year survival rates for lung cancer vary globally but are consistently low (7.5-16%) . Approximately 40% of patients with advanced unresectable disease at the time of diagnosis have a poor prognosis. At present, no single chemo-radiation therapy regimen can be considered standard; despite the treatment choice for unresectable stage III NSCLC, a platinum-based chemotherapy regimen and thoracic radiation are concurrently administered. Chemotherapy concurrently with chest radiation therapy significantly improves the survival of patients with unresectable stage IIIA and IIIB disease. Decades of research have increased understanding the lung cancer as a multistep process involving genetic and epigenetic alterations, through which, resulting DNA damage transforms normal epithelial cells that progressively acquire growth and/or survival advantages until cancer arises [2,4-7]. Malignant transformation of lung epithelial cells is characterized by genetic instability, which can exist at the chromosomal level (with large-scale loss or gain of genomic material, translocations, and microsatellite instability) or at the nucleotide level (with single or several nucleotide base changes). Moreover, lung cancer is also related to genomic and epigenomic changes at the transcriptome (with altered gene and microRNA expression) and proteome [8-11] level. As many kinds of tumors, molecular abnormalities in lung cancer cells are typically targeted to proto-oncogenes, tumor suppressor genes, DNA repair genes, and other genes that can promote outgrowth and immortality of affected cells [12,13]. It is accepted that the successful discovery, validation and implementation of specific molecular markers for early diagnosis, clinical surveillance and determination of tumor response to therapeutic intervention could improve survival rates for patients, but only few biomarkers turned out to be useful in the clinic.
3. Genome biomarkers: The opening to personalized medicine in lung cancer
Nowadays, molecular and genetic studies have shown that some specific molecules contribute to sporadic tumors of lung cancer; they are useful as therapeutic targets and predictive biomarkers . Recently, the National Cancer Institute’s lung cancer mutation consortium (NCI´s LCMC) performed such a study on more than 800 lung adenocarcinoma tumor specimens, examining mutations in established lung cancer driver genes (
Mutated EGFR are present in 10-15% of NSCLC tumors [2,17]. Mutant EGFRs (either by exon 19 deletion or punctual mutation in exon 21 known as L858R) show an increased amount and duration of EGFR activation compared with wild-type receptors . Mutated EGFR can activate RAS/RAF/MEK/MAPK and phosphoinositide 3-kinase (PI3K)/AKT and STAT3/STAT5 pathways [19-21]. Beside the importance of EGFR on lung carcinogenesis, some other molecules have been described as molecular markers for prognosis and therapeutic targets. Gene amplification and mutations in the kinase domain of C-erbB2 (HER-2/neu), a member of EGFR family, have been identified in patients with lung adenocarcinomas with a frequency of less than 5% and 5 to 10% respectively, and its overexpression are involved in ~25% of NSCLC cases . EGFR and HER-2 kinase domain mutations have similar associations with female sex, non-smoking status and Asian background in patients with adenocarcinoma [15,22]. RAS/RAF/MEK/MAPK pathway is involved in signaling downstream from EGFR leading the growth and tumor progression in NSCLC. Activating
Some other molecules have been identified based on expression and genomic data such as MYC and Cyclin D1 which are amplified and over-expressed in 2.5–10% and 5% of NSCLC respectively, while BCL-2 over-expression is involved in ~25% of cases of NSCLC [8,16]. Recent data have shown that methylation of the promoter regions of genes is a common event in NSCLC, which contributes to oncogenes over-expression or tumor genes suppressors silenced. These epigenetic changes may be an early event in NSCLC, since that promoter region of p16 gene is frequently methylated in smokers and premalignant lesion of lung cancer . PI3K-AKT-mTOR pathway is altered in NSCLC. AKT overexpression has been described in a subgroup of NSCLC tumors jointly with mutations or amplification of PIK3CA gene. These genomic modifications are related with enhanced activity of PI3-K pathway mainly in squamous cell carcinoma tumors . On the other hand, tissues of smoker patients show higher levels of angiogenic factors such as VEGF. VEGF expression increases in relationship with tumoral grade, which in turn, correlates with increased microvessel density, development and poor prognosis of lung cancer. Tumoral angiogenesis and angiogenic factors are regulated by hypoxic inductor factor (HIF) 1α and 2α or through oncogenes as
4. Molecular and radiology therapies in lung cancer
4.1. Molecular therapy: Response and biological resistance
EGFR exhibits overexpression or aberrant activation in 50 to 90% of NSCLC. Mutations in EGFR allow sustained activation of EGF signaling for tumor cell survival, therefore, has been development targeted inhibitors for this molecule . mAbs target the extracellular domain of EGFR and small molecules that inhibit intracellular EGFR tyrosine kinase domain function. In 2004, a significant advancement in the treatment of NSCLC was made following the observation that somatic mutations in the kinase domain of EGFR strongly correlated with sensitivity to EGFR TKIs [31, 32]. EGFR mutations are particularly prevalent in a patient subgroups with specific characteristics as adenocarcinoma histology, women, never smokers, and East Asian ethnicity . This subgroup shows an exquisite sensitivity and marked tumor response to TKIs treatment. Despites the results obtained with biological therapies, there is a group of patients who do not respond to molecular therapy. Moreover, there is another group of patients with EGFR mutant lung cancer who initially respond to TKI treatment, but subsequently develop disease progression after a median of 10 to 14 months on treatment with biological therapy [37,38]. Hence, no optimal therapy thereafter has yet been established. Presumably, tumors do not respond because their molecular lesions are downstream of the therapeutic target . Resistance to biologic therapy in NSCLC has been associated with EGFR exon-20 insertions  or a secondary T790M mutation , KRAS mutation , or amplification of the MET proto-oncogene [43,44], where MET is a transmembrane receptor with a tyrosine kinase domain, which activates signaling survival depending to PI3K and MAPK pathways. Of importance, Some reports showed that inhibition of MET signaling can restore sensitivity to TKIs . HER-2 kinase domain mutations are associated with resistance to EGFR TKIs, but also with sensitivity to HER-2-targeted therapy .
Genomics data have provided information for developing targeted therapies in lung cancer patients based upon identification of cancer-specific vulnerabilities and set the stage for molecular biomarkers that provide information on clinical outcome and response to treatment. It has become clear that molecular-targeted cancer therapies can only reach their full potential through appropriate patient selection. In addition, there are now large clinical studies of lung cancer showing distinct chemotherapy and radiation responses. The majority of patients with lung cancer display advanced disease, these patients have obtained modest improvements in overall survival and quality of life through the use of systemic chemotherapy; however, the survival is still low, getting a median survival of 8 to 10 months . Once recurred or metastasized, the disease is essentially incurable with survival rates at 5 years of less than 5%, and this has improved only marginally during the past 25 years . The substantial genetic heterogeneity inherent to human cancers as an indicator of distinct phenotypes makes the identification of patients most likely to benefit from a given anticancer agent challenging. The description of molecules associated with resistance or sensitivity to cytotoxic treatments will improve personalized therapy for lung cancer. Radiotherapy, alone or in combination with surgery or chemotherapy, plays a critical role in the management of lung cancer. More than 60% of lung cancer patients receive radiotherapy at least once during the course of their disease .
4.2. Role of EGFR pathways in resistance and sensibility to radiotherapy
NSCLC tumors exhibit a wide response spectrum to radiation therapy but the molecular basis for this responsiveness is unknown. Some patients with NSCLC have a good response to radiation therapy with long-term local control while others relapse even with high dose treatment . Many factors are involved in biological process of lung damage induced by radiation. At the molecular level, it is established that ionizing radiation causes various types of cellular damage; the creation of DNA breaks represents the principal damage induced by direct action of ionizing radiation or indirect action provoked by reactive species oxygen (ROS). Inadequately repaired DNA breaks leads to loss of cell clonogenicity via the generation of lethal chromosomal aberrations or the direct induction of apoptosis . In addition to DNA breaks, ROS rapidly triggers the production of cytokines, growth factors, and more ROS, ultimately leading to chronic oxidative stress, hypoxia and the nonhealing tissue response in the lung [50,51]. Tumor radioresistance, including intrinsic resistance before treatments and acquired resistance during radiotherapy, is one of the main obstacles for radiotherapy efficiency for NSCLC. Some of the most important mechanisms associated with radioresistance in cancer including checkpoint pathway, mismatch repair process, and DNA damage repair [52-54]. Accumulating evidence suggests that radioresistance is often correlated with some genes, such as p53  and EGFR . In this regard, targeting EGFR pathway activation radiosensitizes human cancer cells [57-59], suggesting that the presence of overexpressed or activated oncogenes such as EGFR or RAS may be a mechanism for increased cellular resistance to radiation. In some models, it has been demonstrated that EGFR/Ras/Raf/MEK/ERK signaling may be activated in response to radiation, promoting cancer cell survival and proliferation [52-54,60] (Figure 3).
Variations in NSCLC responses to radiotherapy alone or in combination with chemotherapy or biological therapy are most likely due in the majority of cases to the genetic and epigenetic constitution of tumors [61,62]. In NSCLC, EGFR and KRAS oncogenes play an important role as prognostic factors; therefore, their role in radioresistance has been documented . NSCLC cell lines harboring EGFR with mutations in tyrosine kinase domain were many folds more sensitive to radiation compared to cell lines with wild type EGFR. Radiosensitivity of NSCLC cell lines with mutant EGFR and human bronchial epithelial cells stably expressing mutant forms of EGFR was attributed to delayed DNA repair kinetics, defective radiation-induced arrest during DNA synthesis or mitosis, and pronounced increases in apoptosis or the occurrence of micronuclei . Apparently, mutant EGFR is unable to translocate into the nucleus, which hinders its interaction with DNA-dependent protein kinase (DNA-PK), which is a fundamental enzyme for repair radiation-induced double strand breaks . Besides of the promising role of mutant EGFR in radiosensitivity, the effort by blocking EGFR pathway to induce better response to radiotherapy has been limited. Inhibition of the EGFR by TKI or mAb, has been shown to
radiosensitize a limited number of NSCLC cell lines
5. Lung cancer radiogenomics
Radiotherapy has played a key role in the control of tumor growth in many cancer patients, including lung cancer. Studies that originated more than 40 years ago [72,73] have indicated that tumors respond to radiotherapy by initiating a process called accelerated repopulation. In this process, the few surviving cells that escaped death after exposure to radiotherapy or chemotherapy can rapidly repopulate the badly damaged tumor by proliferating at a markedly faster pace. This phenomenon suggested that tumoral heterogeneity permits a cell population in the tumor to have advantages to avoid cell death induced by radiation. Cellular senescence, DNA repair and cell cycle checkpoint are cellular mechanisms that influence the resistance to radiotherapy. However, the molecular mechanisms that regulate the radioresistance phenotype have not been clear in cancer. For this reason, some research groups have focused in the study of biological models to obtain genomic and proteomic signatures in order to find genes and proteins that could predict radiosensitivity or radioresistance in lung tumors (Table 1). Although such researches have contributed to a partial understanding of the mechanisms underlying cellular radioresistance, the comprehensive functional mechanisms remain largely elusive. This may be quite reasonable since the mechanisms of radioresistance are a complex multigene interaction. In this sense, Torres-Roca
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The authors developed a radiation classifier to calculate the radiosensitivity of tumor cell lines based on basal gene expression profiles obtained from the literature. They predicted the survival fraction to 2 Gy (SF2) value in 22 of 35 cell lines from the National Cancer Institute, a result significantly different from chance (P = 0.0002). In their approach, radiation sensitivity as a continuous variable, significance analysis of microarrays is used for gene selection, and a multivariate linear regression model is used for radiosensitivity prediction. In gene selection, they identified three novel genes: RbAp48, RGS19, and R5PIA, whose expression values correlated with radiation sensitivity. Exogenous overexpression of RbAp48 into three cancer cell lines (HS-578T, MALME-3M, and MDA-MB-231) induced radiosensitization (1.5- to 2-fold), moreover, higher proportion of transfected cells with RbAp48 were in G2-M phase of the cell cycle (27% versus 5%). Finally, RbAp48 overexpression is correlated with dephosphorylation of Akt, suggesting that RbAp48 might be exerting its effect radiosensitized by antagonizing the Ras pathway, but it could also do so through PI3K. The authors establish that radiation sensitivity can be predicted based on gene expression profiles and they introduce a genomic approach to the identification of novel molecular markers of radiation sensitivity. Despite of results in different tumor cell lines, this work included only four NSCLC cell lines and they were able to predict correct SF2 values for only two of them . So, the study should be performed on a broader panel of NSCLC cell lines. In lung cancer, multiple studies have identified a wide array of genetic and epigenetic alterations, including mutations in DNA sequence, DNA copy number changes, aberrant DNA promoter methylation, changes in mRNA, microRNAs and protein expression , revealing many potential determinants and signaling pathways governing lung tumorigenesis and progression. Gene expression profiling analysis allows for an increase in the understanding of the molecular mechanisms and pathways that involve radioresistance. Thus, the strategy followed by Torres-Roca and collaborators can be applied to gene expression data reported in lung cancer, in order to identify new molecular targets for radiotherapy response. In this sense, we know that the response of tumor cells to radiation is accompanied by complex changes in the gene expression pattern. Based on mRNA expression profiles and systems-biology approach, Eschrich
A problem in radiogenomics research is the difficulty to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. For understand the radiation-mediated changes in gene expression that might result in different responses to radiation, Guo W
researches have been focused in describing specific molecules that revert the radioresistant phenotype. It is well known that there is a large amount of cell death during cytotoxic cancer therapy such as radiotherapy; therefore, radioresistance is associated with deregulation of apoptosis proteins. Sun
6. Proteomics of radiation response in lung cancer
Despite proteomics being useful to find molecular markers associated to lung cancer cells , in radiation resistance research there are very few studies focused on applying proteomics to find new markers associated to radiotherapy response in lung cancer. Recently, Wei R
One of the most important problems in lung oncology is lack of suitable biomarkers as therapeutic targets or the absence of predictors of therapy response. The genetic heterogeneity of the lung tumors influences the initial molecular resistance to therapies, but also in the development of resistance during treatment. The molecular mechanisms that influence the resistance to biological or radiological treatments, referring to the resistance mechanisms occurring naturally because of the carcinogenic process, or those developing as a result of evolutionary pressure that tumor cells undergoing during the treatment administration, is a barrier that has not been fully elucidated. With current genomics and proteomics studies in lung cancer focused on solving the mystery of therapeutic resistance, it has been possible to identify molecules that may serve as prognostic markers of response to radiological and molecular therapy resistance. Genes and proteins that regulate cell proliferation and survival, including signaling molecules and transcription factors such as KRAS, BRAF, PI3K, MAPK, mTOR, JAK2, STAT, survivin and others have demonstrated to be part of the molecular machinery that regulates therapeutic resistance. Moreover, gene and protein expression profiling of lung cancer has focused specifically on searching predictive markers to radiotherapy. Some studies have generated data on molecules involved in radioresistance or radiosensitivity either natural or acquired. Using therapeutic doses of radiation in
Authors gratefully acknowledge the financial support from the National Council of Science and Technology (CONACyT), Mexico (grants 115552 and 115591), and The Institute of Science and Technology (ICyT-DF), Mexico (grant PIUTE147).