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Abstract
Background: Colorectal carcinoma (CRC) is the most common malignancy of the gastrointestinal tract, representing an incredible health problem. It is essential to develop drugs against novel targets––involved in CRC tumorigenesis and progression––to improve the management of the disease. The aim of this study was to evaluate C-X-C chemokine receptor type 4 (CXCR4) and Peroxisome proliferator-activated receptor gamma (PPAR-γ) expression in CRC, and to associate their expression with the available clinicopathological parameters. Materials and Methods: This study included 50 cases of primary CRC. All cases were stained by CXCR4 and PPAR-γ antibodies to assess their immunohistochemical expression. The relations between their expression and clinicopathological variables were assessed. Results: CXCR4 expression was detected in 76% of studied cases. High CXCR4 expression showed significant associations with the depth of tumor invasion (P = 0.024), lymph node metastasis (P = 0.009), advanced tumor stage (P = 0.001) and the presence of vascular invasion (P = 0.035). PPAR-γ expression was detected in 78% of studied cases. PPAR-γ expression showed a statistically significant inverse relation with histologic types (P = 0.001), tumor grade (P = 0.005), depth of tumor invasion (P = 0.001), lymph node status (P = 0.001), TNM stage (P = 0.002), and vascular invasion (P = 0.001). Conclusions: High CXCR4 and decreased PPAR-γ expressions are related to high tumor grade, advanced stage, and vascular invasion in colorectal carcinoma.
Keywords: Colorectal carcinoma, CXCR4, immunohistochemistry, PPAR-γ
How to cite this article: Introduction 
Colorectal carcinoma (CRC) is the most common malignancy of the gastrointestinal tract. It represents the third most common cancer worldwide among men, and the second among women, with about 1.2 million new cases diagnosed yearly.[1] The high mortality of CRC is mainly due to local recurrences and distant metastasis nearly in 75% of cases, mainly to the liver.[2]
Despite the great advances in the diagnosis and treatment of CRC, it remains an incredible health problem. Therefore, it is essential to develop drugs against novel targets––involved in CRC tumorigenesis and progression––to improve detection, prognosis, and treatment of this disease.[3] C-X-C chemokine receptor type 4 (CXCR4) and peroxisome proliferator-activated receptor gamma (PPAR-γ) protein are of these promising therapeutic targets.
CXCR4, encoded on chromosome 2, is a chemokine G-protein–coupled trans-membrane receptor, involved in hematopoietic and immune regulation, wound healing, and inflammation. CXCR4 binds CXCL12 [also named chemokine stromal cell-derived factor-1 (SDF-1)], released by the stromal cells. CXCR4/CXCL12 binding plays an important role in cancer progression by giving malignant phenotype to the tumor microenvironment and triggers various downstream signal transduction cascades.[4]
These downstream pathways lead to elevated intracellular calcium, gene transcription, chemotaxis, angiogenesis, cell proliferation, and survival, also promote therapy resistance, and permits tumor cell metastasis from primary sites to metastatic ones. Therefore, blocking CXCR4 may be a successful target to limit the metastatic potential of CRC.[5]
CXCR4 expression enhances epithelial-to-mesenchyme transition (EMT), in which epithelial cells gradually lose their polarity, membrane adhesion, and cell-to-cell contact, to acquire spindle morphology, therefore favoring migration of the tumor cells. However, the precise molecular mechanisms of the CRCR4-mediated homing of cancer cells to specific metastatic sites remain unclear.[6]
Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a transcription factor that belongs to nuclear hormone receptor superfamily [retinoid X receptor (RXR)] and plays an important role in tissue homeostasis and fatty acid metabolism. PPAR-γ is normally expressed in many cell types throughout the body, mainly the adipocytes, monocytes and macrophages, liver, skeletal muscle, breast, prostate, and colonic epithelial cells.[7]
In cancer cells, PPAR-γ plays an anti-proliferative and pro-differentiation role, yet this role remains unclear. PPAR-γ induces cellular differentiation via up-regulating E-cadherin gene expression. PPAR-γ also Inhibits cell proliferation via several mechanisms; PPAR-γ increases the expression of cyclin-dependent kinase (CDK) inhibitors, p21, p27, and PTEN while decreasing the levels of cyclin D1 and E and nuclear factor NFκB. PPAR-γ also induces apoptosis by up-regulation of Bax and down-regulation of Bcl-2. PPAR-γ can inhibit angiogenesis by decreasing the levels of vascular endothelial growth factor (VEGF) and can decrease the metastatic ability of cancer cells via down-regulation of matrix metalloproteinases (MMPs).[8]
The relation between CXCR4 and PPAR-γ is still largely unexplained. CXCR4 may act as an effector target of PPAR-γ, and its expression could be regulated by ligand-activated PPAR-γ.[9]
This work aimed to study CXCR4 and PPAR-γ expression in CRC, and to associate their expression with each other, and with the available clinicopathological parameters.
Materials and Methods This study was carried out on 50 cases of colorectal carcinoma patients' who underwent radical colectomy without previous chemotherapy or radiation. Formalin-fixed paraffin-embedded blocks (FFPE) were collected retrospectively from the archives of the Pathology Department,Faculty of Medicine, Tanta University, during the period of the research from January 2019 to March 2021. Patients' data, regarding age, sex, and tumor location, were obtained from their medical records. Approval from the Research Ethics Committee (REC), [BLINDED FOR PEER REVIEW], was taken before conducting this study.
H and E examination of the cases was done, to confirm the histopathological diagnosis and to evaluate various histological features including the histopathological grade, depth of invasion (T), lymph node status (N), and vascular invasion. Specimens were classified and graded according to the World Health Organization (WHO) criteria.[10] Staging was determined according to the TNM classification of the American Joint Committee on Cancer (8th edition).[11]
Paraffin-embedded sections were immunostained for CXCR4 and PPAR-γ. After de-waxing, inactivating endogenous peroxidase activity and blocking cross-reactivity with normal serum. Overnight incubation was done in a humidity chamber with a rabbit polyclonal anti-CXCR4 antibody (Clone PA3-305) (Thermo Scientific, Egypt), dilution 1:50 and with a mouse monoclonal anti-PPAR-γ antibody (Clone A3409A) (Thermo Scientific, Egypt), dilution 1:50, followed by washing in phosphate-buffered saline (PBS). Sections were then covered with 4–5 drops of Ultra Vision biotinylated goat anti-polyvalent secondary antibody, incubated at room temperature for 10 minutes, then washed in PBS. Finally, sections were counterstained with Meyer's hematoxylin.
Evaluation of CXCR4 and PPAR-γ immunohistochemical staining
All slides were examined under magnification x400. CXCR4 and PPAR-γ expressions were detected as brownish staining of the cytoplasm and nucleus of the neoplastic cells, respectively. The scoring system was assessed by incorporating both the percentage of stained cells and the intensity of the stain. The percentage of positive stained cells was: 0 (negative), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). Staining intensity was: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong staining). The final score was calculated by multiplying the percentage of the stained cells and the staining intensity.
For CXCR4 expression, cases were classified into two groups: low expression (score 0-3) and high expression (score 4-12).[12]
For PPAR-γ expression, 0 points defined as negative (-), 1-2 points as positive (++), and >2 points as strongly positive (+++). For statistical purposes, PPAR-γ expression was divided into two groups: negative (scores 0-1) and positive expression (scores >1).[13]
Statistical analysis
Statistical analysis was done using Statistical Package for Social Science (SPSS version 23). Data were expressed as frequencies for categorical variables and mean ± SD for the continuous variables. For comparing categorical variables, Chi-square (χ2) was used as the test of significance. P value < 0.05 was considered statistically significant.
Results The clinicopathological characteristics of the studied cases are summarized in [Table 1].
Relation between CXCR4 expression and clinicopathological variables
High CXCR4 expression was detected as brownish cytoplasmic staining of the tumor cells in 38 cases (76%). High CXCR4 expression showed significant associations with the depth of tumor invasion (T) (P value = 0.024), lymph node metastasis (N) (P value = 0.009), advanced tumor stage (P value = 0.001), and the presence of vascular invasion (P value = 0.035).
No significant relation was demonstrated between CXCR4 expression and tumor location, histologic types, and histopathological grade [Table 2] and [Figure 1].
Figure 1: Conventional adenocarcinoma (grade I) showing low CXCR4 cytoplasmic expression, score (0) (x400) (a), Mucinous carcinoma (grade II) showing high CXCR4 cytoplasmic expression, score (6) (x400) (b), Conventional adenocarcinoma (grade III) showing high CXCR4 cytoplasmic expression, score (8) (x400) (c) Conventional adenocarcinoma (grade II) showing high CXCR4 cytoplasmic expression, score (8) (x400) (d), Conventional adenocarcinoma (grade II) showing high CXCR4 cytoplasmic expression, score (12) (x400) (e), Mucinous carcinoma (grade III) showing high CXCR4 cytoplasmic expression, score (12) (x400) (f)Relation between PPAR-γ expression and clinicopathological variables
PPAR-γ expression was detected as brownish nuclear staining in tumor cells of 39 out of the 50 studied cases (78%). There was a statistically significant inverse relation between PPAR-γ expression and histologic types (P value = 0.001), histopathological grade (P value = 0.005), depth of tumor invasion (T) (P value = 0.001), lymph node status (N) (P value = 0.001), TNM stage (P value = 0.002), and vascular invasion (P value = 0.001). No significant association was demonstrated between PPAR-γ and tumor location [Table 3] and [Figure 2].
Table 3: PPAR-γ expression in relation to clinicopathological variables
Figure 2: Conventional adenocarcinoma (grade II) showing positive nuclear PPAR-γ expression score (+8) (x400) (a), Conventional adenocarcinoma (grade III) showing positive nuclear PPAR-γ expression, score (+8) (x400) (b), Conventional adenocarcinoma (grade III) showing negative PPAR-γ expression, score (0) (x400) (c), Mucinous carcinoma (grade III) showing positive nuclear PPAR-γ expression, score (+12) (x400) (d), Mucinous carcinoma (grade III) showing negative PPAR-γ expression, score (0) (x400) (e)Relation between CXCR4 and PPAR -γ expression in the studied CRC cases
A significant relation was detected between CXCR4 and PPAR-γ expressions (P = 0.035). CXCR4 and PPAR-γ co-expression was detected in 28 cases (56%) [Table 4].
Table 4: Relation between CXCR4 and PPAR-γ immunoreactivity in studied cases of colorectal carcinoma Discussion CRC is a worldwide health and economic burden, with high metastatic and death rates. The development of efficient treatment options is necessary to improve the prognosis and prolong the survival of CRC patients. In this work, we studied the expression of CXCR4 and PPAR-γ in CRC and their associations with each other and different clinicopathological parameters.
CXCR4 is a chemokine receptor, expressed at low levels or even absent in normal tissues. CXCR4 is involved in tumor growth and metastasis in various human cancers. Blocking CXCR4 signaling pathway has emerged as a potential therapeutic target for human tumors.[5]
In this study, high CXCR4 expression was detected in 76% of our studied cases. Its expression was significantly associated with the depth of tumor invasion (T), lymph node status (N), advanced tumor stage, and vascular invasion. No significant relation was found between CXCR4 expression and tumor location, histologic types, and histopathological grade.
Our results matched those of Romain et al.,[14] who found that CXCR4 expression was absent in colonic polyps and early-stage carcinomas, and that high CXCR4 expression was related to advanced TNM stages, liver and nodal metastasis, and poor survival of CRC patients'.
Scala[15] and Jiang et al.[16] reported that higher CXCR4 expression was associated with poor tumor differentiation, increased local recurrences, advanced stages, lymph node involvement, liver, and distant metastasis, and decreased overall survival.
Murakami et al.,[17] also reported that high CXCR4 expression was significantly higher in metastatic lymph nodes, and liver foci compared to primary tumors and found that CXCR4 negative tumors showed significantly reduced metastasis to lymph nodes, liver, and lung. They stated that drugs capable of targeting CXCR4 axis could be a promising therapy for CRC treatment and may protect against CRC metastasis.
Yoshuantari et al.[18] did a quantitative evaluation of CXCR4 messenger RNA (mRNA) expression in 32 CRC patients, and found that all tumor tissues showed higher levels of CXCR4 compared to normal colonic tissue. They found an association between CXCR4 expression and lymphatic invasion, but not with the patients' ages, gender, clinical stage, the histological grade, or any recurrences.
In contrast to our results, Du et al.[19] observed no association between CXCR4 expression and pathological stage, lymphovascular invasion, and the 5-year disease-free survival. Also, they reported no relation between CXCR4 expression and patient's sex, age, tumor size, location, preoperative carcinoembryonic antigen, or vascular invasion.
Yu et al.[20] studied the role of CXCR4 in promoting progression of inflammatory CRC through recruiting immunocytes and enhancing cytoskeletal remodeling, which may provide novel therapeutic targets for blocking CXCR4-induced CRC progression.
Benedicto et al.[5] reported that the disruption of the CXCR4/CXCL12 axis by CXCR4 antagonist blocked the liver metastasis of CRC. D'Alterio et al.[21] found an in vitro role of CXCR4 antagonism [using CXCR4 antagonist Peptide R (Pep R)] in potentiating colon cancer standard therapy.
PPAR-γ ligands have anticancer activity against many neoplastic cells in vitro. Yet, it is still unclear if high levels of PPAR-γ is associated with favorite prognosis in cancer patients. The anti-proliferative effects of natural PPAR-γ ligands [as 15-Deoxy-Δ-Prostaglandin J2 (15d-PGJ2)], and its synthetic agonists [as thiazolidinediones (TDZ)] have promising applications in cancer prevention and therapy. PPAR-γ agonists can inhibit cell proliferation and tumor growth induce apoptosis, slow the rate of metastasis and cancer progression, and can be effective in treatment of CRC in preclinical studies.[9]
In the current study, PPAR -γ expression was detected in 78% of the studied cases. There was a statistically significant negative relation between PPAR-γ expression and the histologic types, histopathological grade, depth of tumor invasion (T), lymph node status (N), TNM stage, and vascular invasion.
Our results matched those of Yaghoubizadeh et al.,[22] who found that PPAR-γ was suppressed in CRC tumor tissues compared with the adjacent normal tissues. They found that decreased PPAR-γ expression was associated with advanced tumor stage, grade, size, distant metastasis and declined overall survival rate of CRC patients, suggesting that PPAR-γ may act as a tumor suppressor gene in CRC.
Milone et al.[23] observed that reduced PPAR-γ expression in CRC tissues due to epigenetic silencing is accompanied by CRC progression and patients' worse prognosis. Beyaz et al.,[24] also found evidence that the suppression of PPAR-γ, compromised the immunosurveillance and antitumor function of the effector T cells, through impairing their mitochondrial oxidative metabolism, thereby, resulting in tumor progression.
In contrast to our results, Theocharis et al.[25] found that PPAR-γ positivity was not associated with Dukes' stage, histological grade, lymph node and liver metastasis, vascular invasion, tumor proliferative rate, or patient survival, but it was associated with the expression of cell cycle-related molecules (Rb, cyclin D1, p16, and p21). They suggested the importance of specific PPAR-gamma ligands as cell cycle modulators for a future therapeutic approach in colon cancer.
In the current study, 66.7% of mucinous adenocarcinoma showed negative PPAR-γ expression, compared with 7.9% PPAR-γ negative conventional adenocarcinoma. This was because most mucinous adenocarcinomas in our study were poorly differentiated and presented at higher stages. Park et al.,[26] confirmed this fact and stated that; compared with conventional adenocarcinoma, mucinous adenocarcinoma commonly presented with advanced stages, with an increased risk of metastasis and poor prognosis, as mucin may cause more dissemination of the tumor cells. These results suggest that PPAR-γ is an important factor involved in cancer progression, which might be a potential therapeutic target for treatment of CRC.
Many studies reported the contradictory role of PPAR-γ in cancer, depending on the cell type. PPAR-γ may act as a tumor suppressor gene (in colon, lung, ovarian cancers, and neuroblastoma).[27] Paradoxically, other studies suggested that PPAR-γ ligands may be pro-tumorigenic (in prostatic or bladder cancers).[28]
Niu et al.[29] found that PPAR-γ was significantly down-regulated in epithelial ovarian carcinoma and that there were significant associations between low PPAR-γ expression and advanced FIGO stage, distant metastasis, recurrence, poor overall survival. On the contrary, Elix et al.[30] studied PPAR-γ expression in prostatic carcinoma and found that its level increased with advanced grade and stage, which would suggest that PPAR-γ expression may play a role in prostate cancer progression, not suppression, and that PPAR-γ inhibition might be useful in prostate cancer prevention.
In our work, a statistically significant relation was detected between CXCR4 and PPAR-γ expressions. The relation between CXCR4 and PPAR-γ had been studied by Rovito et al., who stated that CXCR4 acts as an effector target gene of PPAR-γ, and demonstrated that its expression is negatively regulated by ligand-activated PPAR-γ. They found that PPAR-γ was able to reduce cancer cell invasion and motility through CXCR4 down-regulation in colon and lung cancers. However, they stated that the mechanism by which PPAR-γ modulated CXCR4 expression remains largely unclear.[9]
In conclusion, our results suggest that high CXCR4 expression and decreased PPAR-γ expression are related to high tumor grade, advanced stage, and vascular invasion in colorectal carcinoma. We recommend further studies on larger number of cases to reveal the relation between CXCR4 and PPAR-γ, and to determine the therapeutic benefits of targeting CXCR4 and PPAR-γ for treatment of CRC.
Ethics approval and consent to participate
The current study was conducted after obtaining the approval from research ethics committee, Faculty of Medicine, Tanta University, Egypt.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_481_21
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