Background
Head and neck cancer (HNC) is one of the most prevalent cancers worldwide [
1‐
3]. The standard treatment for patients with HNC is surgery, radiation, chemotherapy or a combination of these treatments [
4,
5]. Although treatment strategies have advanced in the last two decades, the overall 5-year survival rate for patients has not significantly changed. Tumor recurrence after radiotherapy is a major obstacle to recovery in HNC [
4,
5]. The identification of radioresistant molecules contributing to a poor prognosis may facilitate patient consulting to determine proper treatment selection to improve the therapeutic outcome.
Previous studies have globally surveyed human genes associated with radioresistance in various cancers. Thus far, different experimental approaches have been used, including the direct comparison of two sets of samples with different levels of radiosensitivity in cancer tissues or cell lines [
6‐
8]. A major disadvantage of this approach lies in the heterogeneity of the samples; the different radiosensitivities among various genetic backgrounds or tissue origins may also have a confounding effect on other pathological phenotypes. The use of microarrays to compare gene expression profiles between parental and radiation-treated cells has also been reported [
9,
10]. Although this approach minimal the cause of genetic variation between individuals, those reports on the cellular and molecular effects often used short-term treatments of irradiation as a research model and may reflect the external radiation-induction situation. To recapitulate the condition of patients with intrinsic radioresistance, we have established several isogenic HNC cell sublines by long-term and low-dose serial irradiation [
11,
12]. These subline cells have been shown possessing higher radioresistant phenotype compared to their parental cells [
11,
12]. Similar approach has also been employed and demonstrated to efficiently generate high radioresistant cells [
13‐
16].
To obtain a more comprehensive profile of the molecular network associated with radioresistance, in this study, we performed cDNA microarrays to globally survey differential transcriptomes between HNC cells and isogenic radioresistant sublines. We also used bioinformatic software to assess core molecular pathways associated with radioresistance. Through data mining of gene expression profiles available in the public domain, we further identified the biomarker signatures that contribute to the prognosis of HNC. Our study provides predictive or prognostic information, which may be further applied as biomarker criteria regarding treatment choices for individualized therapy in HNC.
Discussion
Radiotherapy is an indispensable treatment modality in HNC, while radioresistance is the major cause of treatment failure. Therefore, the identification of resistant molecules may allow further clinical applications in personalized radiotherapy. For this goal, we have obtained several important findings in this study. (1) Without interference by genetic heterogeneity, 255 genes were identified to be associated with radioresistance in HNC cells, including 155 up-regulated and 100 down-regulated genes. (2) Four core molecular pathways significantly contributed to radioresistance in HNC cell lines and HNC patients: modulation of cellular focal adhesion, the PI3K-Akt signaling pathway, HIF-1 signaling pathway, and pluripotency of stem cells. (3) Eleven molecules were associated with a poor survival in HNC, 4 of which, IGF1R, LAMC2, ITGB1, and IL-6, were key molecules to predict a worse prognosis of radiotherapy. Further validation studies are highly recommended to confirm these results in a subgroup of HNC patients, such as for a specific anatomic subsite, a specific ethic patient group, or for the cancers caused by a specific etiology.
Four core pathways played substantial roles related to radioresistance in HNC. In the pathway modulating focal adhesion, 11 RR genes were enriched (Tables
1 and
2). Molecules in the laminin (LAMA3, LAMC2) and integrin (ITGA6, ITGB1, ITGB4) families composed the majority in this pathway, indicating the close link between these signaling pathways and radioresistance. Laminin, a family of extracellular matrix glycoproteins, is the major non-collagenous constituent of the basement membrane. It comprises 3 chains, alpha, beta and gamma, to form various heterotrimeric laminin isoforms [
20]. Integrin is a family of transmembrane receptors that facilitates extracellular matrix adhesion. It consists of alpha and beta subunits with several isoforms to form a heterodimeric protein [
21]. Although the major functions of laminin and integrin are to maintain the mechanical integrity of the cell membrane and regulate cell mobility, recent studies have shown much more biological roles than what was originally thought. Through the interaction of laminin and its cell-surface receptors, including integrin, this complex protein activates signal transduction pathways that may mediate various cellular functions, including cell proliferation, differentiation, invasion, tumor angiogenesis and metastasis [
21‐
23]. In this study, we further noted that up-regulation of this focal adhesion pathway plays an important role in facilitating radioresistance in HNC (Figs.
1 and
2). Our finding is consistent with recent reports that cell adhesion molecules, such as laminin and integrin family proteins, participate in radioresistance, chemoresistance, or cell survival in several cancers [
24‐
28]. We also demonstrated that LAMC2, a laminin component, and ITGB1, an integrin subunit, exhibit excellent correlation with poor outcome in HNC patients receiving radiotherapy (Figs.
3 and
4). These results are supported by other investigations as well. LAMC2 is over-expressed in several types of cancers, including urothelial, lung, colorectal and head-neck cancers, and is associated with a poor prognosis [
29‐
33]. Similarly, a high level of ITGB1 predicts poor survival in breast and gastric cancers [
34‐
38]. Furthermore, LAMC2 or ITGB1 may serve as therapeutic targets via modulating molecular expression to improve radiosensitivity [
39‐
42]. All these results highlight the importance of the laminin-integrin axis pathway in radioresistance and cancer aggressiveness.
Another pathway identified to be associated with radioresistance, the PI3K-Akt signaling pathway, was shown to be highly significant (Figs.
1 and
2). PI3K-Akt is an intracellular signaling pathway that promotes cell growth and survival in response to extracellular stimuli [
43]. It is an important downstream mediator of several membrane-bound receptor tyrosine kinases. Hyper-activation of this pathway correlates with many aggressive cancer phenotypes, such as cell proliferation, tumor angiogenesis, metastasis, and a poor prognosis [
43]. Furthermore, the PI3K-Akt pathway contributes to radioresistance in many types of cancers, a finding that was consistent with ours [
44,
45]. In this study, we also noted that IL-6 is a key extracellular signaling molecule participating in the PI3K-Akt signaling pathway (Tables
1 and
2). IL-6 is a pleiotropic cytokine that is involved in multiple biological responses, including auto-immunity, inflammation and cancers [
46,
47]. It can mediate numerous downstream effectors by activating several signaling cascades, including the JAK/STAT, MAPK, and PI3K/AKT pathways, to promote cancer progression [
47‐
50]. Specifically, the function of IL-6 in therapeutic resistance has been commonly reported. This molecule can protect cells from radiation- or drug-induced DNA damage by suppressing oxidative stress or the induction of anti-apoptotic mechanism to facilitate cell survival [
50‐
53]. Clinically, IL-6 has also been found to be over-expressed in almost all types of tumors and is associated with a poor prognosis of cancers such as cervical, lung, gallbladder, and head-neck cancers [
54‐
58]. All these reports support our findings that IL-6 is a critical factor that is over-expressed in cancers and regulates the PI3K-Akt signaling pathway, leading to radioresistance and a worse treatment outcome.
The pathways of cancer stemness and HIF-1 signaling exhibited a prominent function in this study (Figs.
1 and
2). Recently, accumulating studies have shown that cancer stem cells, a subset of cancer cells, possess stem cell-like properties, which may serve as the driving force for tumorigenesis. These cells have a strong malignant potential, with self-renewal capacity, high mobility, and stress tolerance, resulting in resistance to chemo-radiotherapy [
59,
60]. The HIF-1 signaling pathway is critical in the maintenance of the cancer stemness phenotype, which may be induced by the hypoxia condition in the tumor microenvironment [
61‐
63]. This signaling pathway is also an important factor contributing to radioresistance in many cancers [
64,
65]. All these reports support our findings, suggesting that the intra-tumor hypoxia condition induces the HIF-1 signaling pathway, further facilitating cancer stemness formation and resulting in radioresistance in HNC. We also noted that IGF1R is an important molecule participating in the HIF-1 signaling pathway and regulating cancer stemness (Tables
1 and
2). These results are consistent with previous studies by other investigators [
66‐
69]. IGF1R has tyrosine kinase activity, which plays a significant role during cancer cell transformation mainly via cytoprotection and anti-apoptosis [
70‐
72]. Apparently, high expression of this molecule in tumors confers resistance to chemo-radiotherapy [
70‐
72]. Consistently, we found that IGF1R is over-expressed in cancer tissues and is associated with adverse outcomes in HNC patients receiving radiotherapy (Figs.
3 and
4). Similar results were also found in many types of cancers, including prostate, lung, ovary and renal cancers [
73‐
77]. Taken together, our results demonstrated that IGF1R is an unfavorable prognostic marker in HNC patients treated with radiotherapy. This may result from modulation of the HIF-1 signaling pathway, leading to stem cell conversion and a radioresistant phenotype.