Overexpression of MLPH in Rectal Cancer Patients Correlates with a Poorer Response to Preoperative Chemoradiotherapy and Reduced Patient Survival

Data mining of a public transcriptomic rectal cancer dataset (GSE35452) from the Gene Expression Omnibus, National Center for Biotechnology Information identified the melanophilin (MLPH) gene as the most significant intracellular protein transport-related gene (GO:0006886) associated with a poor response to preoperative chemoradiation. An MLPH immunostain was performed on biopsy specimens from 172 rectal cancer patients receiving preoperative chemoradiation; samples were divided into high- and low-expression groups by H-scores. Subsequently, the correlations between MLPH expression and clinicopathologic features, tumor regression grade, disease-specific survival (DSS), local recurrence-free survival (LRFS), and metastasis-free survival (MeFS) were analyzed. MLPH expression was significantly associated with CEA level (p = 0.001), pre-treatment tumor status (p = 0.022), post-treatment tumor status (p < 0.001), post-treatment nodal status (p < 0.001), vascular invasion (p = 0.028), and tumor regression grade (p < 0.001). After uni- and multi-variable analysis of five-year survival, MLPH expression was still associated with lower DSS (hazard ratio (HR), 10.110; 95% confidence interval (CI), 2.178–46.920; p = 0.003) and MeFS (HR, 5.621; 95% CI, 1.762–17.931; p = 0.004). In conclusion, identifying MLPH expression could help to predict the response to chemoradiation and survival, and aid in personal therapeutic modification.


Introduction
Colorectal cancer remains among the most frequently diagnosed cancers in the world, with significant cancer-associated mortality [1]. On the basis of current evidence, the standard treatment for patients with advanced rectal cancer involves surgery with preoperative radiation with or without chemotherapy [2]. These advanced therapeutic options have improved patient survival. However, 10-20% of patients still experience recurrence or metastasis after initial treatment [3]. Although these rectal cancer patients with tumor failure may be eligible for salvage treatment, they still face a dismal outcome. Therefore, it is important to find reliable genetic biomarkers as predictors of the response to preoperative therapy and prognosis, and to aid in personal therapeutic modification.
The intracellular transport of cellular proteins and organelles is critical for maintaining the structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles [4,5]. The current literature reports that changes in the expression of these proteins have been associated with cancer invasion, metastasis, and survival [6,7]. Therefore, to identify the potential biomarkers related to intracellular protein transport in rectal cancer, a public transcriptomic dataset of rectal cancer (GSE35452) from the Gene Expression Omnibus, National Center for Biotechnology Information (GEO, NCBI, Bethesda, MD, USA) was used and melanophilin (MLPH) was recognized as the most significantly up-regulated gene (GO:0006886).
Therefore, our current study aimed to investigate the association between MLPH expression and clinical outcomes, including tumor response to preoperative chemoradiation and survival, in our 172 rectal cancer patients treated with preoperative chemoradiation.

Data Mining of the Published Transcriptomic Rectal Cancer Dataset
In the NCBI GEO public transcriptomic database (GSE35452), information from 46 rectal cancer patients who had undergone preoperative chemoradiation was used for data mining. To quantify the expression levels, the raw. cel files on the Affymetrix Human Genome U133 Plus 2.0 microarray platform (Affymetrix, Inc., Santa Clana, CA, USA) were analyzed using the Nexus Expression 3 (BioDiscovery, Hawthorne, CA, USA) software, employing a comparative analysis without filtering or preselection. According to the response to preoperative chemoradiation, the samples were separated into "responders" and "nonresponders", with special attention given to the genes involved in intracellular protein transport (GO:0006886). Transcripts with p < 0.01 and an expression fold change > ±0.1 log2 ratio were selected for analysis.

Patients and Tumor Characteristics
In this study, patients with newly diagnosed rectal adenocarcinoma between 1998 and 2004 were identified from the cancer registry database of Chi Mei Medical Center. The clinical staging was based on abdominal computed tomography (CT) or magnetic resonance imaging (MRI), and was revised according to the 7th edition of the American Joint Committee on Cancer (AJCC) staging system. All patients received standardized long-course chemoradiation, including 5-fluorouracil-based chemotherapy either orally or intravenously every week from the commencement of radiation therapy to completion. The radiation dose ranged from 45-50 Gy in 25 fractions and the whole course usually ran over a 5-week period. Radical surgery with total mesorectal excision (abdominoperineal resection or low anterior resection) was performed 4-6 weeks after chemoradiotherapy. Approximately 3 to 4 weeks after surgery, 5-fluorouracil-based adjuvant chemotherapy was administered for at least 4 months if the pre-treatment or post-treatment tumor or nodal status was beyond T3 or N1, respectively. Patients with incomplete clinicopathological information or a history of either cancer or metastatic disease were excluded. Finally, a total of 172 rectal cancer patients who were regularly followed up on until death or the final follow-up were included, and their formalin-fixed paraffin-embedded (FFPE) tissue specimens were used for analysis. The data extracted from the medical records included the date of diagnosis, age, gender, clinical/pathological characteristics (such as location, CEA level, stage, lymphovascular invasion, perineural invasion, and chemotherapy use), and cause of death, if relevant.

Histopathologic and Immunohistochemical Analysis
Tumor FFPE specimens were evaluated by a pathologist blinded to the patients' clinical information. The extent of tumor regression (tumor regression grade, TRG) after preoperative chemoradiation was assessed using a five-point system, and the study patients were classified as having: a poor response with TRG 0-1 (<25% response); a moderate response (TRG 2-3); or no visible tumor in the rectal wall with TRG 4 (complete response) [8]. As our previous studies described, the method of immunohistochemistry included deparaffinized, rehydrated, heated, quenched, and washed for pretreatment rectal tumor biopsy specimens [9,10]. Subsequently, the tissue sections were incubated for 1 h with a primary antibody recognizing MLPH (ThermoFisher Scientific, Waltham, MA, USA; Clone: OTI6E3, 1:200). The H-score was calculated to interpret the MLPH protein expression and was quantified using the following equation: H-score = Σpi (I + 1), where Pi is the percentage of stained tumor cells for each intensity (0-100%), and i represents the intensity of staining (0 to 3+) [11]. The definition of high and low MLPH expression were as follows: high expression, above or equal to the median; and low expression, below the median of all scored cases.

Statistical Analysis
The chi-square test was used to determine the association between MLPH expression and clinicopathological characteristics. The Kaplan-Meier method was applied to estimate our endpoints: the 5-year disease-specific survival (DSS), local recurrence-free survival (LRFS), and metastasis-free survival (MeFS), with the log-rank test used for comparison. Recurrence or deaths due to cancer were defined as events. All factors were analyzed using univariate Cox regression to estimate hazard ratios (HRs) with 95% confidence intervals (CIs). Those factors with statistical significance were entered into multivariate analysis. All statistical analyses and graphics were executed using SPSS for Windows 22.0 (IBM Corporation, Armonk, NY, USA). A p value less than 0.05 was determined to be statistically significant.

Up-Regulation of the MLPH Gene Is the Most Significant Intracellular Protein Transport Factor Related to Preoperative Chemoradiotherapy
As described above, a public transcriptomic dataset (GSE35452) that included 46 rectal cancer patients was analyzed to find potential biomarkers, focusing on intracellular protein transport (GO: 0006886). As shown in Table 1 and Figure 1, MLPH was the most significantly up-regulated gene associated with a poor response to preoperative chemoradiotherapy (log 2 ratio, 1.2845; p < 0.0001). This finding prompted an investigation of the relationship between MLPH expression and clinicopathologic behavior in rectal cancers after chemoradiation. Table 2 details the characteristics of the 172 patients with rectal adenocarcinoma treated with preoperative chemoradiotherapy followed by radical surgery. Most of these patients were men (108, 62.8%) and the median age was 63 years (range, 22-88 years). AJCC tumor staging identified 41.9% of patients at stage I, 29.9% at stage II, and 28.1% at stage III disease. Patients with clinical or pathological stage II or III disease received adjuvant chemotherapy. In terms of other pathological features, 15 (8.7%) patients presented with vascular invasion and 5 (2.9%) with perineurial invasion. After chemoradiation, 17 (10%) patients had complete tumor regression (TRG 4), 118 (68.6%) patients had a moderate response (TRG 2-3), and 37 (21.5%) patients had a poor response (TRG 0-1). In the Supplementary Figure S1, TRG was statistically significantly correlated with 5-year DSS, LRFS, and MeFS. For patients with a complete response (TRG 4), the 5-year DSS was higher than TRG 2+3 and TRG 0+1 (92% versus 76% versus 42%, p < 0.05). They were also statistically significant in 5-year LRFS and MeFS (p < 0.05).

Immunohistochemical Analysis
After dividing our rectal cancer patients into groups with high and low MLPH expression, immunohistochemical staining was performed to assess the relationship of this factor with clinicopathologic characteristics (Figure 2). Cytoplasmic expression of MLPH was successfully scored in all examined cases with a wide range of H-scores, varying from 100 to 290. As shown in Table 2, high MLPH expression was significantly associated with CEA ≥ 5 (p = 0.001), pre-treatment tumor status (T3-4; p = 0.022), post-treatment tumor status (T3-4; p < 0.001), post-treatment nodal status (N1-2; p < 0.001), vascular invasion (p = 0.028), and poor response to preoperative chemoradiotherapy (p < 0.001). In the group with high MLPH expression, we also observed a tumor regression grade of 0-1 in 30 (17.4%) patients, grade 2-3 in 51 (29.7%) patients, and grade 4 in 5 (2.9%) patients, indicating that MLPH expression plays an important role in the aggressive behavior of rectal tumors and their sensitivity to chemoradiation.

Prognostic Significance of MLPH Expression
In Table 3, the univariate analysis indicated that clinicopathological variables, such as post-treatment tumor status and TRG, were significantly associated with a worse DSS, LRFS, and MeFS rate (all p ≤ 0.05). Location from anal verge was significantly associated with DSS (0.0473), and pre-treatment nodal status was significantly only related to LRFS (p = 0.007). CEA level (≥5 ng/mL) and presence of vascular invasion were negatively associated with DSS and LRFS to statistical significance (p ≤ 05 for all). Most importantly, rectal cancer patients with MLPH overexpression had significantly lower DSS (p < 0.0001), LRFS (p = 0.0002), and MeFS (p < 0.0001), as shown in Table 3 and Figure 3. In multivariate analyses (

Discussion
Previous studies have revealed that MLPH is associated with tumor development, progression, and metastasis in many cancer types [12][13][14][15]. However, its role in rectal cancer, especially in terms of the response to chemoradiation, remains unknown. By considering the predisposing clinicopathological factors, our study observed that MLPH overexpression is associated with advanced tumor status, poor response to chemoradiation, and worse survival. These findings indicate that MLPH may be a potential biomarker to predict survival and a promising therapeutic target in the treatment of rectal cancer.
Tumor response after preoperative chemoradiation in rectal cancer varies considerably. Previous studies have demonstrated that down-staging or even the complete disappearance of the malignant cells in the rectal wall and perirectal nodes is associated with better survival [8,16]. Therefore, an accurate evaluation of the tumor response to preoperative chemoradiation is essential for predicting clinical outcomes and developing further treatment plans. Many techniques are used to evaluate the response after preoperative treatment: endorectal ultrasounds, CT scans, MRIs, or positron emission tomography scans [17][18][19][20]. However, these imaging systems cannot provide accurate information on the stage of the tumor (T or N categories) when they are compared with preoperative images. An alternative method of evaluation includes assessing levels of carcinoembriogenic antigen or other molecular markers [21][22][23]. No conclusion has yet been reached as to which factor or marker (e.g., Ki-67, p21, p53, or Cox-2) is representative of cancer progress, due to the small sample size of the majority of these studies [24][25][26]. Thus, we sought to provide a better marker for disease progression.
Data mining of a public transcriptomic rectal cancer dataset (GSE35452) from the NCBI GEO identified the MLPH gene as the most significantly intracellular protein transportrelated gene (GO:0006886) associated with poor response to preoperative chemoradiotherapy. The MLPH gene is known to encode a member of the exophilin subfamily of Rab effector proteins [27]. The protein forms a ternary complex with the small Ras-related GTPase Rab27A in its GTP-bound form and the motor protein myosin Va [28]. MLPH is an important component of the melanosome transport mechanism, which is involved in the visible pigmentation of hair and skin. Diseases associated with MLPH include Griscelli syndrome type 3, which is characterized by a silver-gray hair color and abnormal pigment distribution in the hair shaft [29].
In recent decades, MLPH had been found to be associated with cancer. Many studies have demonstrated that MLPH contributes to tumor development, behavior, and metastasis in skin, breast, and prostate cancer [14,15,30]. Using The Human Protein Atlas database, Guo et al. found that MLPH expression was significantly up-regulated in malignant melanomas compared with benign nevi and normal skin [30]. Moreover, moderate to high melanophilin expression has been commonly observed in patients with advanced-stage melanoma. In breast cancer, Thakkar et al. found MLPH gene expression in aggressive tumors (basal-like subtypes) [14]. Another study noted downregulation of the MLPH gene in lymph node-positive breast cancer patients [12]. Zhang et al. also observed that MLPH expression is related to cell migration, proliferation, and invasion in prostate cancer [15]. All of these studies support our findings that higher MLPH expression is significantly associated with advanced tumor behavior, including pre-treatment tumor status, posttreatment tumor status, post-treatment nodal status, and vascular invasion. Therefore, altered MLPH biology is likely to play an important role in colorectal tumor formation and progression.
Although the association between MLPH expression and colorectal cancer outcomes remains unknown, our study is the first to demonstrate that a high expression of MLPH is associated with a poor response to preoperative chemoradiation and confers a negative impact on survival (DSS, LRFS, and MeFS). Similarly, Guo et al. observed that MLPH expression is altered in melanoma patients, and high levels of MLPH expression are associated with poor survival (p = 0.02), based on data from The Cancer Genomics Atlas database [30]. Zhang et al. similarly observed a decreasing trend in the overall survival of the patients with prostate cancer who had a high expression of MLPH [15]. Collectively, these data indicate that high MLPH expression is not only associated with a more aggressive cancer phenotype, but also acts as a predictor of poor prognosis.
More importantly, we found that high MLPH expression was also significantly associated with lower TRG scores, indicating a poor response to preoperative chemoradiation. The possible mechanisms for this association are as follows. First, transforming growth factor beta (TGF-β) is a key downstream effector of MLPH [31]. TGF-β is known to modulate late post-radiation changes, such as DNA repair, cell cycle progression, inflammation at an early stage, and the later development of fibrosis [32]. Aberrant MLPH expression is associated with an inhibition of the regulation of TGF-β and the decreased production of profibrotic growth factors, leading to a poor response to radiation [33]. Second, MLPH also acts as an effector protein for Rab27, which has long been implicated in the progression of various cancers. Recent studies have reported that Rab27 plays a significant role in the resistance to chemotherapy or radiation in many types of cancer [34,35]. Thus, MLPH expression is involved in the resistance to radiotherapy or chemotherapy, leading to lower odds of survival.
There are some limitations to this study that need to be addressed. This study was analyzed using a public transcriptomic dataset and tumor tissues from our cohort. We did not verify the MLPH mRNA or protein levels through PCR or Western blotting, which may further support our findings regarding chemoradiation therapy. Second, we included some early-stage rectal cancer patients who were receiving preoperative chemoradiation for organ preservation. Therefore, we were able to comprehensively observe the association between MLPH expression and early-/advanced-stage groups and their tumor regression grade. Finally, our study lacks information on factors such as mesorectal fascia involvement, KRAS, BRAF, and MMR status of the tumors. Future studies that include these factors are necessary to confirm our results.

Conclusions
This study is the first to reveal that MLPH expression can be a prognostic marker for rectal cancer outcomes. Our results indicate that high levels of MLPH expression are associated with significantly aggressive tumor behavior, poor response to chemoradiation, and shorter survival. Therefore, further study to better understand the molecular mechanisms of MLPH could provide effective insights for rectal cancer treatment. Studies should also be undertaken to determine whether MLPH expression could serve as an effective marker of prognosis in order to better guide clinical treatment plans.