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ORIGINAL ARTICLE
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Significance of cancer testis-associated antigens (SPAG9 and FBXO39) in colon cancer


1 Department of Chemical Pathology, Medical Research Institute, Alexandria University, Egypt
2 Department of Human Genetics, Medical Research Institute, Alexandria University, Egypt
3 Department of Experimental and Clinical Surgery, Medical Research Institute, Alexandria University, Egypt
4 Department of Internal Medicine, Medical Research Institute, Alexandria University, Egypt
5 Alexandria University Students' Hospital, Egypt

Date of Submission02-Oct-2019
Date of Decision05-Feb-2020
Date of Acceptance21-May-2020
Date of Web Publication11-May-2021

Correspondence Address:
Ragaa A Ramadan,
Department of Chemical Pathology, Medical Research Institute, Alexandria University
Egypt
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijc.IJC_873_19

  Abstract 


Background: Cancer testis antigens (CTA) are normally expressed in immune privileged tissues such as the testis. They are considered tumor-associated antigens because they are specifically expressed in different cancers. Their distinct nature rendered them appealing targets for cancer diagnosis, prognosis. and immunotherapy. We aimed to identify the association of two CTA genes with colon cancer (CC) in a cohort of Egyptian patients.
Methods: We measured the relative gene expression levels of two CTAs: SPAG9 and FBXO39 in colonic tumor tissue and adjacent normal-appearing mucosa in 50 newly diagnosed colon cancer patients by real-time reverse transcription polymerase chain reaction. Gene expression was also studied in relation to demographic and pathological criteria.
Results: SPAG9 and FBXO39 were overexpressed in 22% and 40% of cases, respectively. Overexpression of both genes was evident in 14% of cases. We report the significant expression of FBXO39 (P < 0.01) in tumor tissue compared to normal tissue. SPAG9 was significantly increased in large sized tumors compared to smaller sized tumors. Otherwise, there was no significant association between gene expression and the evaluated clinicopathological features (P > 0.05).
Conclusions: SPAG9 and FBXO39 are possible CC diagnostic biomarkers. Further studies are warranted to validate our findings.


Keywords: Cancer testis antigens, colon cancer, gene expression, immunotherapy, real time reverse transcription polymerase chain reaction
Key Message
In the era of precision medicine, the gene expression of cancer-testis antigens as SPAG9 and FBXO39 can be a useful diagnostic marker in the management of colon cancer.



How to cite this URL:
Ramadan RA, Morsy H, Samir M, Shamseya M, Shalaby M, El-Attar EA. Significance of cancer testis-associated antigens (SPAG9 and FBXO39) in colon cancer. Indian J Cancer [Epub ahead of print] [cited 2021 Jun 13]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=315810





  Introduction Top


Cancer cells usually express specific proteins that can trigger a humoral immune response against the tumor and are thus named tumor-associated antigens. They include several subclasses one of which is the cancer testis antigens (CTAs), which are exclusively expressed in normal testicular tissue and trophoblasts, as well as some tumors. The precise mechanism of action of CTA is somehow obscure but they contribute significantly to spermatogenesis and fertilization.[1]

CTAs have been previously linked to tumorigenesis with postulation of the carcinogenic transformation of dormant embryonic remnants. This theory was supported by the shared features between gametogenesis and oncogenesis. In addition, some tumors originate from displaced trophoblasts and germ cells. Embryo/germ cell-related proteins such as alpha-fetoprotein and human chorionic gonadotrophin are now routine tumor markers.[2]

Molecular studies of CTA genes revealed inconsistency and different frequencies of expression, whether by real-time reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemical assay.[3] Sperm-associated antigen 9 (SPAG9), located at chromosome 17q21.23, encodes a leucine zipper dimerization. It acts as a scaffolding protein in the c-Jun N-terminal kinase (JNK) signaling pathway, which mediates several physiological processes. It has a specific expression in the acrosomal compartment of the sperm and participates in spermatozoa–egg interaction.[4] Its expression whether as mRNA or protein was successfully demonstrated in several tumors such as lung and prostate cancer.[5],[6] F-box proteins are integral parts in the substrate-recognition subunits of SKP1– cullin 1–F-box protein (SCF) type E3 ubiquitin ligase complex. They interact in various cellular functions, for instance, immune responses. However, their main function is the ubiquitylation and consequent degradation of target proteins.[7] FBXO-39 is a member of F-box proteins located on 17p31.1[8] whose overexpression was demonstrated in different cancer cell lines.[9] The expression of these two CTAs in malignancy rendered them a possible point of research especially that they were not fully explored in colon cancer (CC).

Worldwide, colorectal cancer (CRC) is the third most commonly diagnosed cancer after lung and prostate in men, and second in women.[10] In Egypt, it ranges from 2.1–2.9% in men and 2.08–2.31% in women.[11] It results from the interaction of genetic, epigenetic, and environmental factors. Our research team has been looking for significant genes suitable for the diagnosis and prognosis for CRC in the Egyptian population.[12]

Although surgery, chemotherapy, and radiotherapy are employed as the main treatment modalities, tumor recurrence and chemoresistance are still frequently encountered. Hence, researches focus on discovering markers for early detection along with new lines of cancer treatment. Clinical trials questioned the significance of immune-based therapeutic approaches namely immunomodulatory monoclonal antibodies (immune checkpoint inhibitors), adoptive cell transfer, and cancer vaccines.[13],[14] Identification of targets for immunotherapy has been the current focus in research not only for treatment but also for prevention in high-risk subjects.[15] The restricted expression of CTA in immune-privileged tissues, as well as their cancer-specific immune response, rendered them appealing targets for cancer diagnosis and immunotherapeutic approaches.

We aimed to study the relative expression of two CTA genes in colonic neoplastic tissue and determine its relation to clinicopathological criteria. Our choice of the genes considered their expression in other solid tumors, CRC cell lines, and their potentials as immunotherapeutic targets.


  Materials and Methods Top


Fifty paired primary CC and adjacent noncancerous colonic tissue samples were obtained from consecutive CC patients who underwent colonoscopy in the endoscopy units of Medical Research Institute, Alexandria University in Egypt from January 2017 to January 2018.

All patients were newly diagnosed and not genetically related. Colonic tissue samples were obtained by colonoscopy prior to any therapy; chemotherapy or radiotherapy treatment. Preoperative staging was based on complete colonoscopy and multidetector computed tomography (MDCT) of the abdomen and pelvis. Tumor staging and grading were done according to the seventh edition of the The Union for International Cancer Control (UICC)/TNM system from surgical biopsies by two independent pathologists. Left-sided tumor refers to those from descending and sigmoid colon, while right-sided includes the cecum, ascending and transverse colon. Exclusion criteria included inflammatory gastrointestinal diseases (ulcerative colitis, Crohn's disease), rectal cancer, and other malignancies. Management protocol at the Alexandria University was set according to National Comprehensive Cancer Network (NCCN) guidelines v2.2018.[16] All our cases were treated by surgical resection irrespective of stage. Patients with stage III and IV received fluoropyrimidine–oxaliplatin combination therapy for 6 months after surgery.

Demographic data were obtained through interviews and standardized questionnaires. Family history of cancer was specified as self-reported CRC in first-degree relatives. A thorough clinical examination and computed tomography of the abdomen and pelvis were performed in all patients. All procedures performed in our study including the informed consent were in accordance with the ethical standards of the Ethical Committee of Medical Research Institute, Alexandria University (IORG#: IORG008812) and with the 1964 Declaration of Helsinki and its later amendments.


  Methods Top


Molecular study of relative gene expression includes the following steps

Tissue collection & homogenization

Biopsies were taken from the core of the colonic tumor mass and from adjacent nontumor tissue (matched macroscopically normal-appearing mucosa) at least 15 cm away from the suspicious lesion. Fresh punch biopsies were immediately and completely submerged in the preservative, RNAlater stabilization reagent (Qiagen, Germany) in sterile Eppendorfs. They were incubated in the refrigerator at 4°C for 24 hours to allow full permeation of the preservative into the tissue, then transferred to −80°C freezer until tissue processing. Later, thawed tissue biopsies were first weighed and adjusted to 30 mg for each sample. Tissue homogenization was achieved mechanically. Ribonucleic acid (RNA) lysis buffer was added while grinding to ensure proper disruption of cells.

Total RNA extraction

SV Total RNA Isolation System (Promega, USA, Cat #Z3100) was used according to the manufacturer's instructions. Total RNA concentration in the extract was determined by NanoDrop™ 1000 Spectrophotometer (Thermo Scientific, USA). The ratio of absorbances A260/A280 and A260/A230 were used to indicate the purity of the RNA extract. Elutes were stored at −80°C.

Reverse transcription

Reverse transcription was done using RevertAid First Strand cDNA synthesis reagents (Thermo Scientific, USA, # K1622) according to manufacturer's protocol. Then, the relative quantitation of the genes was performed by quantitative polymerase chain reaction (qPCR) (Eco illumine, USA) using Maxima SYBR Green qPCR master mix (Thermo Scientific, USA, # K0251). Quantification was done in duplicates. Target gene expression was normalized to the reference gene beta-actin (ACTB). The used primers were validated by primer-BLAST program provided by the National Center for Biotechnology Information (NCBI)[17] [Table 1]. The thermal profile was initial denaturation at 95°C for 10 minutes, followed by 40 cycles with denaturation at 95°C for 15 seconds, annealing/extension at 60°C for 60 seconds. No template control (NTC) consisting of H2O was included in each run. RT-PCR products were also electrophoresed on 2% agarose gel to check product sizes and specificity. Finally, the relative expression of genes was calculated using the comparative cycle threshold method (Livak method).[18]
Table 1: Sequences of primers

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Statistical analysis

We performed a study power calculation. Using NCSS/PASS 2000 software, a sample size of 50 biopsies from tumor tissue and 50 biopsies from adjacent nontumor tissue achieved 95% power for detection of FBXO39 and 90% for SPAG9. Kolmogorov–Smirnov test of normality revealed significance in the distribution of some variables, so nonparametric statistics was adopted. Wilcoxon signed-rank test was used to compare the gene expression between tumor and adjacent nontumor tissue. Comparisons were carried out between groups using Mann–Whitney U test. Nonparametric Kendall's tau correlation (τ) was used for correlation analysis between both genes.


  Results Top


The study included 50 CC cases (median age 57, range 23–80 years), 31 (62%) were women and 19 (38%) were men (ratio 1.6:1). No family history of CRC or inflammatory bowel disease was reported [Table 2]. Adenocarcinoma was the dominant type presenting 48 (96%) cases. A total of 33 (66%) were left-sided tumors and the main site was the sigmoid occurring in 21 (42%) patients. Stage III was most prevalent in our subjects 35 (70%) [Table 3].
Table 2: Clinicopathological characteristics of colon cancer patients

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Table 3: Tumor related variables of colon cancer patients

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We interpreted the relative expression of CTA genes in two ways. First by using the Wilcoxon signed-rank test. The median values of the relative expression of both genes were higher in tumor than the adjacent nontumor tissue, but only FBXO39 reached the level of statistical significance (P < 0.01) [Table 4]. Second, we defined mRNA overexpression as 3-fold higher expression than the median expression in adjacent nontumor tissue. SPAG9 was overexpressed in 11 (22%) cases and FBXO39 was overexpressed in 20 (40%) cases. About 7 (14%) cases showed overexpression of both genes.
Table 4: Relative gene expression levels of SPAG9 and FBXO39 in normal and tumor tissue

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Regarding the relation with clinicopathological features, SPAG9 showed a significant increase in large-sized tumors compared to smaller size tumors. Otherwise, there was no significant association between expression of the genes with the remaining evaluated clinicopathological features including age, gender, staging, and metastasis (P > 0.05) [Table 5]. Furthermore, we performed a correlation analysis between both genes and there was no correlation between SPAG9 and FBXO39 gene expression.
Table 5: Association of SPAG9 and FBXO39 relative gene expression and clinicopathological variables

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  Discussion Top


CRC presents a high economic burden owing to its high mortality and morbidity. Early detection and discovery of better diagnostic molecular biomarkers are needed. In this study, we evaluated the relative expression of two CTAs (SPAG9 and FBXO39) in CC. FBXO39 showed significantly higher expression levels in tumor versus adjacent nontumor tissue, as well as a significant association of SPAG9 with large-sized tumors. Literature review reveals the substantial variation of CTA genes expression which can be attributed to a lack of uniformity in the study protocol, analytical methodology, and interpretation of gene expression.[1]

We report overexpression of SPAG9 in 22% of cases. However, when using Wilcoxon signed-rank test increased expression in tumor compared to tumor-free tissue did not reach the level of statistical significance. Meanwhile, cancer research demonstrated the clinical significance of SPAG9 expression in a variety of tumors such as bladder[19] and prostate carcinoma.[20] Moreover, serum levels of SPAG9 showed a favorable diagnostic performance for detecting endometrial cancer.[21],[22] The role of SPAG9 in tumorigenesis may be explained through its binding affinity to the members of mitogen-activated protein kinase (MPK) family and the subsequent regulation of JNK-signaling cascade, thus activating matrix metalloproteinases and modulating cancer cell proliferation, invasion, and chemoresistance.[4],[23]

Tumor size is considered the main determinant of TNM classification in a number of solid tumors such as breast, lung, and thyroid cancers. On the other hand, it was overlooked in CC to the degree of being excluded from the TNM staging.This claim was raised by Saha et al.[24], who used data from the American National Cancer Data Base (NCDB). They confirmed the assumption that tumor size is directly related to the TNM stage. The larger the tumor size, the higher the TNM stage, and the lower the 5-year overall survival.[24] However, another study concluded that smaller sized tumors have poor survival. But, this assumption was stage-dependent.[25] In our work, SPAG9 raised an alarming sign of tumor invasiveness by the significant increase in large-sized tumors compared to smaller size ones, which denotes a possible prognostic value. Other studies also reported the association of SPAG9 with tumor aggressiveness and TNM staging in the lung[5] and prostate cancer.[6]

In CRC, Kanojia et al.,[26] reported SPAG9 expression in the majority of cases and is correlated with the humoral immune response in early-stage tumors. They suggested that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. In accordance with our findings, Tavakoli et al.,[27] found elevated expression of the SPAG9 gene in approximately 66% of their CRC tissues, as compared to adjacent noncancerous tissues but this did not reach the level of statistical significance. Meanwhile, they could not report any significant relation between gene expression patterns and any clinicopathological criteria. Interestingly, in-vitro downregulation of SPAG9 showed promising results of inhibition of cancer proliferation and invasion in CRC[26] and breast cancer.[28] Hence, the role of SPAG 9 in targeted therapy is emphasized.

FBXO proteins are integral components of the SCF (Skp1CUL-1-F-box protein) type E3 ubiquitin ligase complex entailed in identification, recruitment, and degradation of substrates for ubiquitination, thereby ensuring normal cellular activities.[7] A number of FBXO proteins facilitate the ubiquitin-mediated degradation of cancer-related proteins (like p53 and BRCA1) and they display oncogenic or tumor-suppressive functions.[29] Furthermore, experimental evidence denotes that conformational structural changes of FBXO proteins induce cell apoptosis and withhold cells in the G1 phase.[30]

In 2011, Song et al.[31] introduced the FBXO39 gene as a new CT antigen, that was expressed in CC tissues. In this study, FBXO39 expression was significantly overexpressed in tumor tissue compared to adjacent noncancerous tissue. Similarly, FBXO39 was upregulated in a few tumors such as invasive breast carcinoma.[32] In 2016, Tarnowski et al.[33] used quantitative RT-PCR, with a methodology very similar to our workflow, to assess the expression of a number of CT antigens in CRC. They noted that 6 of 18 (33%) CT antigens tested; DKKL1, MAGEA3, TTK, PLU1, OIP5, and FBXO39 were significantly overexpressed in tumor tissue compared with normal tissue. However, the overexpression of FBXO39 in cancer tissue was not associated with disease progression. Our work confirms the results concluded from their study that FBXO39 was overexpressed in CC tumor tissue but could not be correlated to any of the clinicopathological data of the patients.

In addition, another study by Motalebzadeh et al.[34] reported the significant expression of FBXO39 in CRC tissue compared to normal tissue. FBXO39 gene expression was significantly related to lymph node status. There was no significant association with the remaining clinicopathological variables. Worth mentioning, they noticed the expression of FBXO39 in their three CRC cases of stage 0. But it was difficult to deduce a statistical conclusion from such a small sample size. Our study could not confirm such findings as we only had two patients in our cohort with carcinoma in-situ; one exhibiting an overexpressed FBXO39 gene while the other did not. Nevertheless, our results along with others strongly suggest FBXO39 as a possible diagnostic biomarker for CC. Recently FBXO39 knockdown was shown to significantly inhibit proliferation and induce apoptosis of U2OS osteosarcoma cells. Thus, the role of FBXO39 may be extended from diagnosis to targeted therapy.[35]

Several studies examined the presence of panels of CTA genes and denoted the co-expression of more than one CT gene in a number of tumors with variable frequency.[33],[36] In our study, co-expression of both studied genes was found in 14% of cases.

Mounting evidence supports the concept that cancer is categorized as a stem cell disease originating from the activation of pluripotent cancer stem cells.[37] In this context, CTA expression has been associated with malignant transformation of normal stem cells.[38],[39],[40] Therefore, our perception of CTAs is not confined to their diagnostic potentials suggested in our study, but it is extended to their role in new therapeutic modalities. Oncology clinical trials are focusing on the combined use of CTAs with conventional chemotherapy and DNA methyltransferase inhibitors through novel immunotherapeutics such as checkpoint blockade and cancer stem cell targeting therapy.[41] CTAs can modulate the side effects of treatments including autoimmune reactions and tolerance. Such synergistic approaches could be the hope in the era of personalized medicine to improve mortality and morbidity associated with CC.

As with other recent study designs, tackling gene expression in CC,[42],[43],[44] we compared tumor to tumor free-tissue in the same patient from biopsies taken before any surgical intervention. We excluded other gastrointestinal inflammatory diseases such as ulcerative colitis and Crohn's disease. Despite the possibility of the presence of some degree of microscopic non-specific colitis in the tumor environment or in tumor-free tissue, FBXO39 expression was able to differentiate between tumor and nontumor tissue in the same patient.

In conclusion, our results support the probable diagnostic value of FBXO39 expression and point to the possible association of SPAG9 with tumor size. Further follow-up studies are warranted to question the role of CTA in prognosis and patient survival.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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