Overexpression of the MRE11-RAD50-NBS1 (MRN) complex in rectal cancer correlates with poor response to neoadjuvant radiotherapy and prognosis

Colorectal cancer (CRC) is one of the most prevalent malignancies. Although CRC patient mortality rates are decreasing because of improved screening and treatment methods, CRC remains the third most commonly diagnosed cancer in the United States [1] and the third most important cause of cancer-related death globally [2]. Colonic and rectal cancers are often combined as a single entity in many studies but they differ in their metastatic pattern, drug response, and optimal treatment methods [3]. Rectal cancer patients experience poorer survival outcomes than colon cancer patients because resection is more difficult [4]; therefore, selection of appropriate treatment is especially important for rectal cancer patients.

Neoadjuvant (i.e., preoperative) radiotherapy or chemoradiation is routinely used to treat patients with rectal cancer to improve surgical outcomes [3]. Many studies have found that this therapy improves overall survival (OS) and reduce recurrence [5, 6]. Meta-analyses have shown that neoadjuvant radiotherapy improves local control [7], prevents recurrence [8], and reduces mortality [9], but the effect on a given outcome varies greatly between studies. This inconsistency is due in part to the large variation in outcomes between patients. Only 10–30% of patients show a complete response to preoperative chemoradiation, and 70% show a decrease in the tumor stage [10, 11]. Neoadjuvant radiotherapy also has a significant risk of adverse effects in rectal cancer patients, especially gastrointestinal disorders such as bowel obstruction, abdominal pain, and nausea [12]. Although these adverse effects have become less prevalent as irradiation techniques have improved [13], it remains important to select patients carefully for radiotherapy to avoid unnecessary side effects.

Many studies have sought biomarkers that would predict the patient’s response to radiotherapy or chemoradiotherapy, including imaging findings, gene mutations, and expression levels of mRNAs and proteins [14]. The radiosensitivity index (RSI) is a 10-gene signature that predicts the response to radiotherapy in cell lines [15], and has been shown to predict OS in glioblastoma patients [16]. A radioresistance (RadR) score calculated based on expression of 13 genes was associated with recurrence in head and neck squamous cell carcinoma patients treated with adjuvant radiotherapy [17]. More than 40 potential molecular biomarkers have been assessed for their ability to predict outcomes in rectal cancer patients receiving neoadjuvant therapy, often with conflicting results [14]. Therefore, there is still a great need for simple, accurate biomarkers that will predict rectal cancer patient outcomes.

DNA damage repair has been described as a “double-edged sword” in cancer [18]. Defective repair can lead to genome instability and promote cancer formation. Deficiencies in the mismatch repair (MMR) pathway, for example, cause a hypermutability phenotype known as microsatellite instability (MSI). This can lead to overall genetic instability and mutations in many other genes that promote cancer development and progression [19]. MSI is found in approximately 15% of rectal cancers [20]. Conversely, DNA damage is also the mechanism by which radiotherapy and some chemotherapy treatments cause cancer cell death. Thus, cancer cells that can efficiently repair the damage may become resistant to such therapies. The link between DNA damage repair and radiotherapy makes DNA damage-related proteins attractive targets for developing new therapies and for identifying markers of sensitivity to existing therapies. For example, the levels of phosphorylated DNA damage repair related proteins ATM and γH2AX have been identified as biomarkers for radiosensitivity to ¹²C⁶⁺ radiation in various tumor cell lines [21].

Radiotherapy causes double-stranded breaks (DSBs) in DNA. The highly conserved MRN complex comprises the MRE11, RAD50, and NBS1 proteins, and is one of the first factors to sense and bind DSBs. The MRN complex can physically tether DNA ends together, and also plays an enzymatic role in DNA repair via the nuclease activity of MRE11 [22]. The cell cycle checkpoint kinase ATM is recruited to DSBs and activated with the help of MRN, and ATM then phosphorylates all three MRN subunits, demonstrating a role of the complex in cell cycle progression following DNA damage [23]. These roles of the MRN complex led us to hypothesize that tumors deficient in the MRN complex may be more sensitive to the DNA-damaging effects of radiotherapy. We have previously shown that the combined expression of two protein markers of MRE11 and ATM may be predictive of patient outcomes in rectal cancer [24]. We have also found that postoperative expression of RAD50 correlates to patient outcomes in rectal cancer [25]. In the current paper, we have extended our studies to include NBS1 and investigated whether the combined expression of the MRN complex proteins MRE11-RAD50-NBS1 may be superior in predicting patient outcomes after radiotherapy, and therefore useful for selecting the patients who would benefit from preoperative radiotherapy.

Although neoadjuvant chemoradiotherapy has been shown to improve outcomes over surgery alone [5‐9], responses are variable between individuals and difficult to predict [10, 11]. Methods for predicting response would enable better treatment decisions to be made. The TRG score, which reflects early response to treatments such as radiotherapy, is significantly associated with late response outcomes including recurrence and survival [8‐10]. However, there is a need for accurate, reliable biomarkers of tumor radiosensitivity, to enable better treatment decisions before any therapy is administered. This could avoid unnecessary adverse effects [12, 13] in patients who are unlikely to benefit from radiotherapy.

Efficient DNA damage detection, signaling, and repair after radiotherapy can protect tumor cells against damage. Additionally, avoidance of apoptosis or cell cycle arrest can also allow tumor cells to proliferate even after accumulating DNA damage from radiotherapy. These processes can prolong tumor cell survival and promote poor clinical outcomes. Thus, DNA damage-related proteins are potential biomarkers of tumor radiosensitivity. The ten genes in the RSI are not directly involved in DSB repair, but are often closely connected to DSB repair in functional networks [15]. Of the dozens of molecules studied that might predict survival after preoperative therapy in colorectal cancer patients, many are involved in apoptosis, cell cycle regulation, and DNA damage [14]. Because of its well-known roles in apoptosis and in linking genetic stability to the cell cycle, the association of tumor suppressor p53 with treatment outcomes has been extensively explored; however, these studies have had very inconsistent findings [14]. p21, which is a target of p53, has also been implicated as a potential biomarker. In patients with unresectable rectal cancer treated with preoperative chemoradiotherapy, p21 expression was associated with worse survival, even when adjusted for tumor response [29]. Further studies are required to identify and confirm reliable radiosensitivity biomarkers, but DNA repair proteins remain attractive targets.

Here, we established a biomarker panel comprising the three proteins of the MRN complex, MRE11, RAD50 and NBS1. To compare our previous study that identified the combined expression of MRE11 and ATM as a prognostic biomarker [24], the combined MRN expression had high sensitivity and specificity in samples taken from both the TC and TP. The sensitivity, specificity, and overall accuracy were higher for the combined MRN expression panel than for combined MRE11/ATM expression, both in the TC and TP. We found that high expression of the three MRN complex proteins in the TC was significantly associated with DFS and OS in rectal cancer patients. Rectal cancer patients with high expression of all three MRN proteins are twice as likely to have a poorer DFS (HR = 2.114, 95% CI 1.096–4.078, P = 0.026) and four times more likely to have poor OS (HR = 4.196, 95% CI 0.968–18.191, P = 0.045) outcomes. Interestingly, none of the other clinicopathologic variables were significantly associated with combined MRN expression. Therefore, this panel appears to be specifically prognostic of DFS and OS. When examining the subset of patients who received preoperative radiotherapy, the association between combined MRN expression and outcome remained significant. It is tempting to hypothesise that the prognostic value of this panel may be related to tumor radiosensitivity, and future research is warranted in a larger definitive cohort.

Interestingly, high MRN protein levels are associated with better outcomes in some other cancer types. In early breast cancer, patients with high MRN complex expression experienced the greatest reduction in recurrence from radiotherapy [30]. In two different studies of bladder cancer patients, high MRE11 expression was associated with better cancer-specific survival in patients who underwent radiotherapy rather than a cystectomy [31, 32]. Therefore, the MRN complex may play a very different role in cancers arising from different tissues. Alternatively, the prognostic value of the MRN complex expression may be dependent on certain combinations of chemotherapy, radiotherapy, and surgery, which vary between the treatment modalities preferred for different cancers.

When patients were classified by LN involvement, the association of combined MRN complex protein expression with DFS and OS was observed in LN-positive patients but not LN-negative patients. The value of analyzing LN involvement to predict outcomes has been established. In a study of rectal cancer patients undergoing long-course neoadjuvant chemoradiotherapy, combining LN involvement with tumor grade was prognostic for survival after treatment [33]. It is feasible that some biomarkers may specifically predict outcomes in patients with LN involvement or those without. Quintanal-Villalonga et al. [34] found that a mutated version of the FGFR4 gene was associated with OS only in LN-involved patients. Our findings suggest that the potential prognostic value of the MRN expression panel may be related to the LN involvement of the patient.

One mechanism that could lead to altered expression of the MRN genes is defective MMR. Giannini et al. [35] found that the MRE11 gene was mutated in MMR-deficient tumors and cell lines, but not in those with normal MMR function. All of the tumors we tested expressed the two MMR proteins most frequently mutated in MMR-deficient patients, MLH1 and MSH2. The absence of MSH6 or PMS2 protein expression was not significantly associated with combined MRN expression, but this analysis was limited by the very small number of cases lacking expression of either of these proteins. Therefore, the mechanism underlying the prognostic change in MRN expression identified here seems to be independent of the MMR pathway, and is a subject for further study.

The primary limitation of this study was the inability to analyze the relationship of combined MRN expression with tumor regression response. Only 10.6% of patients were classified as responders to radiotherapy, represented by a TRG score of 0 or 1. Because increased MRN protein expression was associated with worse outcomes in rectal cancer patients, reducing MRN protein expression or activity may possibly sensitize tumors to radiotherapy. MRN complex inhibitors, including mirin and telomelysin, have great radiosensitizing effects in preclinical studies [36‐38]. Telomelysin is undergoing Phase I and II trials for use in patients with melanoma (NCT03190824), esophageal cancer (NCT03213054), and hepatocellular carcinoma (NCT02293850). The high expression of MRN complex consitituents could be a predictor for poor prognosis and chemoresistance in gastric cancer [39]. An adenovirus targeting RAD50 also showed promise in sensitizing nasopharyngeal carcinoma cells to radiotherapy [40]. Alternatively, in patients with higher MRN expression who are expected to have worse outcomes, additional radiosensitizing treatments could be used in combination with neoadjuvant radiotherapy. Heat treatment, for example, shows good radiosensitizing effects in cells and is being explored in cancer patients [41]. Dynlacht et al. [42] found that heat radiosensitization was dependent on a functioning MRE11 protein, further suggesting the utility of this treatment in high MRN expression tumors.

The combined expression of the three MRN complex subunits was significantly associated with OS and DFS in rectal cancer patients, including those treated with neoadjuvant radiotherapy. The findings in this study support the proposal that high tissue expression levels of the three MRN complex proteins are prognostic indicators in rectal cancer and in response to preoperative therapy. The association of MRN proteins with radiosensitivity also suggests that the three MRN complex proteins could be targets for the future development of radiosensitizing agents.