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Colorectal cancer (CRC) is the third most common type of cancer, with a worldwide incidence of about 24.8% in 2020. In Egypt; CRC ranked tenth in males (5.8%) and ninth in females (6.2%) [
Colorectal cancer is a genetically heterogeneous disease in which several and different molecular pathways are involved in transformation of normal colonic epithelium to malignant cells, tumor proliferation and invasion. The expanding knowledge of the molecular pathology of colon cancer resulted in classification of CRC into characteristic molecular subtypes based on gene expression in both tumor cells and infiltrating non tumorous stromal cells, to increase the accuracy of prognosis and design personalized therapies for better management of patients [
Testes-specific protease 50 (TSP50) gene, a member of cancer/testis antigens, encodes for a threonine protease enzyme which was first discovered in breast cancer cells. It is expressed in the normal spermatocytes of testes and is related to spermatogenesis [
Disruption of calcium homeostasis becomes evident as an important step in the processes of tumorigenesis and metastasis through upregulation of genes’ expression which are involved in cellular proliferation, angiogenesis and invasion [
Colorectal carcinoma has been found to express a variety of chemokines, including the multifunctional cytokine interleukin-8 (IL-8), a member of the CXC chemokine family of inflammatory cytokines that stimulates the migration of distinct subsets of cells [
The expression of TSP50 as a potential oncoprotein in association with SERCA2 and inflammatory cytokine (IL-8) in colorectal adenoma (CRA) and CRC and the relation of these biomarkers’ expression to intratumoral inflammatory infiltrate and other clinicopathological and prognostic factors in CRC have not been clarified. Thus, the aim of this work is to study the expression of TSP50, SERCA2 and IL-8 in CRA and CRC to find the relation of these biomarkers’ expression to intratumoral inflammatory infiltrate and other clinicopathological and prognostic factors.
After the approval of local ethical committee, Faculty of Medicine, Tanta University (34340/12/20), 107 selected specimens of CRA and primary CRC (29 CRA and 78 CRC) along with 12 normal colonic mucosal tissues obtained from non-specific colonic lesions, were retrospectively collected from the archive of pathology department and private labs. The specimens were either colonoscopic biopsies (for CRA) or colectomies (for CRC). None of colorectal carcinoma cases had received neoadjuvant chemotherapy.
H&E stained sections were revised to confirm the diagnosis and to assess the grade of dysplasia of CRA, the degree of tumor differentiation, lympho-vascular invasion (LVI) and the intra-tumoral inflammatory cell infiltrate score of CRC. The dysplasia of CRA was graded according to a two-tier grading system into a low and high-grade dysplasia (LGD and HGD respectively) [
For immunohistochemistry, paraffin blocks of embedded tissues were cut into 5-μm-thick sections, deparaffinized in two changes xylene, rehydrated in two changes of alcohol then rinsed in distilled water. Antigen retrieval was performed by first preheating the staining dish containing citrate buffer (10 mM citric acid, 0.05% Tween20, pH 6.0) until temperature reaches 95–100°C then sections were incubated in it for 20–40 min. Sections were incubated overnight at 4°C, to increase the affinity of the antibodies used to their antigens, with the following primary antibodies: anti-TSP50 polyclonal (1:25; ABclonal, United States), anti-SERCA2 polyoclonal (1:100; ABclonal, United States) and anti-IL-8 polyclonal (1:100; ABclonal, United States). The slides were then counter stained with hematoxylin. Breast carcinoma was used as a positive control for TSP50. For IL-8 and SERCA2, infiltrating neutrophils were used as an internal positive control.
Immunohistochemical evaluation of TSP50 protein cytoplasmic staining was performed using semi-quantitative scoring method by multiplying the intensity of staining and the percentage of positive tumor cells. For staining intensity, the sections were scored as 0 (negative), 1 (mild), 2 (moderate) and 3 (strong), and for the percentages: 0 (0–10%), 1 (10–30%), 2 (31–50%), 3 (51–70%) and 4 (71–100%). The final scores were graded as follows: − (0); + (1–3); ++ (4–8); +++ (9–12). The sections were classified into two groups: low expression (−,+); and high expression (++,+++) for statistical analysis [
For SERCA2, the specimens were considered positive if >5% of the colonic epithelial cells showed cytoplasmic and/or plasma membrane staining. Semiquantitative scores were used for SERCA2 stains according to the percentage of positively stained cells (+1: <25%; +2: 25–50%; +3: 51–75%; and +4: >75%). Tumors showing expression scores of ++, +++, or ++++ were considered as the high expression group, whereas those with scores of + or negative staining were regarded as the low expression group [
Cytoplasmic and membranous staining for IL-8 were evaluated. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentage of stained cells was grouped as: no staining = 0, 1–10% of stained cells = 1, 11–50% = 2, 51–80% = 3, and 81–100% = 4. Values of <4 and ≥4 divide the patients into low and high IL-8 expression groups respectively [
Statistical analysis was conducted using SPSS (version 20) (Chicago, IL, United States). The association between clinicopathological factors and the expression of biomarkers was performed using Chi‐square (
For the 29 CRA cases, the age of patients ranged from 40–62 years with a mean age of (42.3 ± 2.08). Eighteen (62.1%) cases were males and the other eleven (37.9%) cases were females. Twenty four adenomas were located in the colon (16 in the left side and 8 in the right side) and five adenomas (17.2%) in the rectum. Thirteen (44.8%) adenomas were of tubular type and the remaining 16 (55.2%) showed tubulovillous configuration. LGD was detected in 15 (51.7%) and HGD in 14 (48.3%) CRA cases
Clinicopathologic characteristics of CRA cases.
Factor | CRA n (%) |
---|---|
Age | |
Mean ± SD | 42.3 ± 2.08 |
<45 | 6 (20.7) |
≥45 | 23 (79.3) |
Sex | |
Male | 18 (62.1) |
Female | 11 (37.9) |
Anatomical site | |
Colon | |
Right side | 8 (27.6) |
Left side | 16 (55.2) |
Rectum | 5 (17.2) |
Histological type | |
Tubular | 13 (44.8) |
Tubulovillous | 16 (55.2) |
Dysplasia | |
LGD | 15 (51.7) |
HGD | 14 (48.3) |
LGD, low-grade dysplasia; HGD, high-grade dysplasia.
A total of 78 CRC cases were studied, the age of patients was ranged from 42–72 years with a mean age of (60.4 ± 7.41), 48 (61.5%) cases were males and 30 (38.5%) cases were females. Twenty (25.6%) tumors were located in the right colon, 46 (59%) tumors were in the left colon and 12 (15.4%) tumors were in the rectum. Most of the cases were adenocarcinoma NOS type (64.1%). Moderate and poor degrees of differentiation were detected in 38 (48.7%) and 22 (28.2%) carcinomas respectively, while well differentiated carcinomas were found in 18 (23.1%) specimens. LVI was found in 45 tumors (57.7%). For TNM staging, T1 tumors were identified in 11 (14.1%) cases, T2 in 53 (68%) cases and T3 in 14 (17.9%) cases. The regional lymph node invasion was recognized in 65 cases (83.3%) and distant metastasis was found in 30 (38.5%) cases. For intra-tumoral inflammation, high ITL and ITN scores were encountered in 40 (51.3%) and 38 (48.7%) tumors respectively
Clinicopathological characteristics of CRC cases.
Clinicopathologic characteristics | n (%) |
---|---|
Age | |
Mean ± SD | 60.4 ± 7.41 |
<45 | 18 (23.1) |
≥45 | 60 (76.9) |
Sex | |
Male | 48 (61.5) |
Female | 30 (38.5) |
Anatomical site | |
Colon | |
Right side | 20 (25.6) |
Left side | 46 (59) |
Rectum | 12 (15.4) |
Histological type | |
Adenocarcinoma NOS | 50 (64.1) |
Mucinous | 18 (23.1) |
Signet ring | 10 (12.8) |
Grade | |
I | 18 (23.1) |
II | 38 (48.7) |
III | 22 (28.2) |
LVI | |
Absent | 33 (42.3) |
Present | 45 (57.7) |
TNM Staging | |
T | |
T1 | 11 (14.1) |
T2 | 53 (68) |
T3 | 14 (17.9) |
N | |
N0 | 13 (16.7) |
N1 | 49 (62.8) |
N2 | 16 (20.5) |
M | |
M0 | 48 (61.5) |
M1 | 30 (38.5) |
ITL | |
Low | 34 (43.6) |
High | 44 (56.4) |
ITN | |
Low | 40 (51.3) |
High | 38 (48.7) |
LVI, lympho-vascular invasion; T, primary tumor size; N, regional lymph node spread; M, distant metastasis; ITL, intra-tumoral lymphocytes; ITN, intra-tumoral neutrophils.
High TSP50 expression was observed in 15 adenomas and it was significantly identified more in CRAs with tubulovillous architecture (68.8%) compared to tubular ones (30.8%) and in adenomas HGD (71.4%) than adenomas with LGD (33.3%) (
Expression of TSP50, SERCA2 and IL-8 in CRA
Factor | TSP50 | SERCA2 | IL-8 | ||||||
---|---|---|---|---|---|---|---|---|---|
Low |
High |
|
Low |
High |
|
Low |
High |
|
|
Histological type | |||||||||
Tubular | 9 (69.2) | 4 (30.8) | 4.144/0.042* | 8 (61.5) | 5 (38.5) | 0.388/0.534 | 10 (77) | 3 (23) | 1.357/0.244 |
Tubulovillous | 5 (31.3) | 11 (68.8) | 8 (50) | 8 (50) | 9 (56.3) | 7 (43.7) | |||
Location | |||||||||
Colonic | 11 (45.8) | 13 (54.2) | 0.333/0.564 | 12 (50) | 12 (50) | 1.506/0.220 | 15 (62.5) | 9 (37.5) | 0.561/0.454 |
Rectal | 3 (60) | 2 (40) | 4 (80) | 1 (20) | 4 (80) | 1 (20) | |||
Dysplasia | |||||||||
LGD | 10 (66.7) | 5 (33.3) | 4.209/0.040* | 11 (73.3) | 4 (26.7) | 4.144 | 13 (86.7) | 2 (13.3) | 6.152/0.013* |
HGD | 4 (28.6) | 10 (71.4) | 5 (35.7) | 9 (64.3) | 0.042* | 6 (42.9) | 8 (57.1) |
*
LGD, low grade dysplasia; HGD, high grade dysplasia; CRA, colorectal adenoma.
Immunohistochemical expression of TSP50 showing:
Higher SERCA2 immunolabelling was detected in tubulovillous (50%) more than tubular adenomas (38.5%) and in CRAs with HGD more than those with LGD (64.3 and 26.7% respectively). Significant statistical relation was recognized between high SERCA2 expression and the degree of dysplasia (
Immunohistochemical expression of SERCA2 showing:
IL-8 high expression was recognized in 10 CRA whereas low expression was detected in 19 CRA. No significant statistical relation was found between IL-8 expression and adenoma type or location (
Immunohistochemical expression of IL-8 showing:
High TSP50 expression was found in 47/78 cases and it was significantly associated with the presence of LVI (73.3%,
Expression of TSP50, SERCA2 and IL-8 in neoplastic cells of CRC.
Factor | TSP50 | SERCA2 | IL-8 | ||||||
---|---|---|---|---|---|---|---|---|---|
Low |
High |
|
Low |
High |
|
Low |
High |
|
|
Anatomical site | |||||||||
Colon | |||||||||
Right side | 12 (60) | 8 (40) | 0.022/0.882 | 9 (45) | 11 (55) | 1.131/0.288 | 6 (30) | 14 (70) | 0.021/0.884 |
Left side | 14 (30.4) | 32 (69.6) | 24 (52.2) | 22 (47.2) | 23 (50) | 23 (50) | |||
Rectum | 5 (41.7) | 7 (58.3) | 4 (33.3) | 8 (66.7) | 5 (41.7) | 7 (58.3) | |||
Histological type | |||||||||
Adenocarcinoma NOS | 16 (32) | 34 (68) | 5.241/0.073 | 23 (46) | 27 (54) | 0.739/0.691 | 17 (34) | 33 (66) | 5.213/0.074 |
Mucinous | 8 (44.4) | 10 (55.6) | 10 (55.6) | 8 (44.4) | 11 (61.1) | 7 (38.9) | |||
Signet ring | 7 (70) | 3 (30) | 4 (40) | 6 (60) | 6 (60) | 4 (40) | |||
Grade | |||||||||
I | 8 (44.4) | 10 (55.6) | 4.028/0.133 | 13 (72.2) | 5 (27.7) | 8.022/0.018* | 14 (77.8) | 4 (22.2) | 11.253/0.004* |
II | 11 (29) | 27 (71) | 18 (47.4) | 20 (52.6) | 12 (31.6) | 26 (68.4) | |||
III | 12 (54.5) | 10 (45.5) | 6 (27.3) | 16 (72.7) | 8 (36.4) | 14 (63.6) | |||
LVI | |||||||||
Absent | 19 (57.6) | 14 (42.4) | 7.595/0.006* | 21 (63.6) | 12 (36.4) | 6.021/0.014* | 23 (69.7) | 10 (30.3) | 15.855/0.0001* |
Present | 12 (26.7) | 33 (73.3) | 16 (35.6) | 29 (64.4) | 11 (24.4) | 34 (75.6) | |||
TNM Staging | |||||||||
T | |||||||||
T1 | 9 (81.8) | 2 (18.2) | 9.601/0.008* | 10 (90.9) | 1 (9.1) | 12.234/0.002* | 8 (72.7) | 3 (27.3) | 8.749/0.013* |
T2 | 18 (34) | 35 (66) | 24 (45.3) | 29 (54.7) | 24 (45.3) | 29 (54.7) | |||
T3 | 4 (28.6) | 10 (71.4) | 3 (21.4) | 11 (78.6) | 2 (14.3) | 12 (85.7) | |||
N | |||||||||
N0 | 12 (92.3) | 1 (7.7) | 18.157/0.0001* | 10 (77) | 3 (23) | 9.860/0.007* | 11 (84.6) | 2 (15.4) | 11.609/0.003* |
N1 | 15 (30.6) | 34 (69.4) | 24 (49) | 25 (51) | 19 (38.8) | 30 (61.2) | |||
N2 | 4 (25) | 12 (75) | 3 (18.8) | 13 (81.2) | 4 (25) | 12 (75) | |||
M | |||||||||
M0 | 28 (58.3) | 20 (41.7) | 18.009/0.0001* | 30 (62.5) | 18 (37.5) | 11.358/0.001* | 26 (54.2) | 22 (45.8) | 5.678/0.017* |
M1 | 3 (10) | 27 (90) | 7 (23.3) | 23 (76.7) | 8 (26.7) | 22 (73.3) | |||
ITL | |||||||||
Low | 20 (58.8) | 14 (41.2) | 9.162/0.002* | 19 (55.9) | 15 (44.1) | 1.725/0.189 | 22 (64.7) | 12 (35.3) | 10.930/0.001* |
High | 11 (25) | 33 (75) | 18 (41) | 26 (59) | 12 (27.3) | 32 (72.7) | |||
ITN | |||||||||
Low | 22 (55) | 18 (45) | 7.908/0.005* | 29 (72.5) | 11 (27.5) | 20.686/0.0001* | 24 (60) | 16 (40) | 8.992/0.003* |
High | 9 (23.7) | 29 (76.3) | 8 (21.1) | 30 (78.9) | 10 (26.3) | 28 (73.7) |
*
LVI, lympho-vascular invasion; T, primary tumor size; N, regional lymph node spread; M, distant metastasis; ITL, intra-tumoral lymphocytes; ITN, intra-tumoral neutrophils.
SERCA2 cytoplasmic overexpression in neoplastic cells was identified in only 5 grade I tumors (27.7%), most of the grade II tumors (52.6%) and in the majority of grade III tumors (72.7%). High SERCA2 expression was found more frequently among tumors with LVI (64.4%) than those without (36.4%). Moreover, SERCA2 overexpression was linked with advanced TNM staging. Tumors with high ITN infiltration (78.9%) showed higher SERCA2 protein expression than those with low ITN infiltration (27.5%) or ITL (either low-or high, 44.1 and 59% respectively) infiltration. Significant statistical association was revealed between high SERCA2 protein expression and tumor grade (
High IL-8 immunoreactivity was identified in 44/78 tumors. This high IL-5 protein expression was significantly detected in moderately differentiated (68.4%) and poorly differentiated (63.6%) carcinomas compared to well differentiated ones (22.2%) (
Multivariable analysis of independent prognostic factors: anatomical site, histological type, tumor grade, LVI (absent versus present), TN staging, intra-tumoral inflammatory infiltrate (low versus high), expression of TSP50, SERCA2 and IL-8 proteins (low expression versus high) was conducted showing that TSP50, SERCA2 and IL-8 overexpression were significantly associated with adverse prognosis along with high tumor grade, LVI, advanced T stage, presence of regional lymph node invasion and high ITL
Multivariable analysis of factors affecting prognosis in colorectal carcinoma cases.
Metastasis | Multivariable |
||
---|---|---|---|
Anatomical site | |||
Colon | |||
Right side | 7/20 | 35% | 0.451 |
Left side | 17/46 | 36.9% | |
Rectum | 6/12 | 50% | |
Histological type | |||
Adenocarcinoma NOS | 20/50 | 40% | 0.656 |
Mucinous | 4/18 | 22.2% | |
Signet ring | 6/10 | 60% | |
Grade | |||
I | 0/18 | 0% | <0.0001* |
II | 18/38 | 47.4% | |
III | 12/22 | 54.5% | |
LVI | |||
Absent | 6/33 | 18.2% | 0.001* |
Present | 24/45 | 53.3% | |
T | |||
T1 | 0/11 | 0% | 0.004* |
T2 | 22/53 | 41.5% | |
T3 | 8/14 | 57.1% | |
N | |||
N0 | 0/13 | 0% | 0.025* |
N1 | 23/49 | 46.9% | |
N2 | 7/16 | 43.8% | |
ITL | |||
Low | 11/34 | 32.4% | 0.026* |
High | 19/44 | 43.2 | |
ITN | |||
Low | 16/40 | 40% | 0.778 |
High | 14/38 | 36.8% | |
TSP50 | |||
Low | 3/31 | 9.7% | <0.0001* |
High | 27/47 | 51.1% | |
SERCA2 | |||
Low | 7/37 | 18.9% | 0.001* |
High | 23/41 | 56.1% | |
IL-8 | |||
Low | 8/34 | 23.5% | 0.017* |
High | 22/44 | 50% |
*
LVI, lympho-vascular invasion; T, primary tumor size; N, regional lymph node spread; ITL, intra-tumoral lymphocytes; ITN, intra-tumoral neutrophils.
A significant positive correlation was found between TSP50 expression on one side and SERCA2 and IL-8 expression on the other side. As 32 (78%) and 37 (84.1%) tumors of high SERCA2 and IL-8 immunoreactivity respectively showed TSP50 overexpression as well (r = 0.383 and 0.554,
Correlation between TSP50, SERCA2 and IL-8 expression in the neoplastic cells of CRC.
TSP50 | r |
|
SERCA2 | r |
|
|||
---|---|---|---|---|---|---|---|---|
Low n (%) | High n (%) | Low n (%) | High n (%) | |||||
SERCA2 | ||||||||
Low | 22 (59.5) | 15 (40.5) | 0.383 | 0.001* | - | - | - | - |
High | 9 (22) | 32 (78) | - | - | - | - | ||
IL-8 | ||||||||
Low | 24 (70.6) | 10 (29.4) | 0.554 | <0.0001* | 26 (76.4) | 8 (23.5) | 0.511 | <0.0001* |
High | 7 (15.9) | 37 (84.1) | 11 (25) | 33 (75) |
*
r, correlation.
TSP50 expression was variable, being low in colorectal normal epithelium (
Expression of TSP50, SERCA2 and IL-8 in normal tissues, CRA and CRC cases.
Factor | TSP50 | SERCA2 | IL-8 | ||||||
---|---|---|---|---|---|---|---|---|---|
Low | High | x2/ |
Low | High | x2/ |
Low | High | x2/ |
|
Normal | 12 (100) | 0 (0) | 15.133/0.001* | 12 (100) | 0 (0) | 11.598/0.003* | 12 (100) | 0 (0) | 15.718/<0.0001* |
CRA | 14 (48.3) | 15 (51.7) | 16 (55.2) | 13 (44.8) | 19 (65.5) | 10 (34.5) | |||
CRC | 31 (39.7) | 47 (60.3 | 37 (47.4) | 41 (52.6) | 34 (43.6) | 44 (56.4) |
*
CRA, colorectal adenoma; CRC, colorectal carcinoma.
Colorectal carcinoma still remains a challenging disease due to several contributing factors: a variety of molecular transformation pathways other than the traditional APC, KRAS and tumor suppressor gene p53 mutations are implicated in CRC development [
Therefore, the aim of this study is to investigate the roles of TSP50 as an oncoprotein with altered calcium metabolism and inflammatory cytokine namely SERCA2 and IL-8 respectively in CRA and CRC, understanding their roles in CRC initiation and propagation and the relation of these biomarkers’ expression to the intratumoral inflammatory infiltrate and other clinicopathological and prognostic factors.
As far as we know, this is the first study that correlates the immunohistochemical detection of TSP50, SERCA2 and IL-8 in CRA and CRC with clinicopathological factors.
Testes-specific protease 50 (TSP50) is a unique member of cancer/testis antigens, with exception of testes, it is not expressed in normal tissues. It is activated and re-expressed in human cancers such as breast cancer and other cancers [
In the present study the expression of TSP50 was examined in CRA, CRC as well as in normal colonic mucosa. It was found that TSP50 was not detected or weakly expressed in the normal mucosa tissues. Whereas TSP50 expression scores were significantly varied between CRA and CRC lesions. The significant increase of TSP50 expression found in CRC and CRA compared to normal mucosal tissues is of great value for considering TSP50 as a crucial protein for the process of tumorigenesis.
Also, the TSP50 expression in CRA neoplastic cells was tested in relation to clinicopathological parameters. It was found that high TSP50 expression was significantly identified in adenomas with tubulovillous architecture more than tubular adenomas and in adenomas with HGD more than those with LGD.
Regarding the TSP50 expression in CRC malignant cells and its relation to clinicopathological and prognostic factors, TSP50 overexpression in epithelial cells was observed more abundantly in moderately differentiated CRC than poorly differentiated tumors with no significant association between high TSP50 expression and tumor grade. On the other hand, TSP50 overexpression in CRC was significantly associated with advanced TNM staging and presence of LVI. Additionally, TSP50 protein upregulation was shown to be significantly associated with high intra-tumoral inflammatory infiltrate (both lymphocytes and neutrophils).
The findings of the present work supported the role of TSP50 in tumor cellular proliferation, migration and invasion reported by previous study which found that TSP50 mutation has led to diminished tumorigenic effect of TSP50 with subsequent decrease in
The interesting finding in the present work was the paradoxical low TSP50 epithelial expression in 54.5% of poorly differentiated CRC including the signet ring cell type and its high expression in associated non-neoplastic stromal cells; which may be attributed to the dual role that TSP50 plays in both epithelial neoplastic cells and stromal cells. Its epithelial expression enhances tumor cellular proliferation and migration, while the stromal expression of TSP50 facilitates the neoplastic epithelial cell invasion through its threonine protease activity. This could explain the heterogeneity of TSP50 epithelial and stromal expression in high grade CRC in the current work. Also, TSP50 expression was found to be variable depending on tumor type, genesis and microenvironment [
Intracellular Ca2+ is kept in a state of equilibrium and regulated by Ca2+ signaling and Ca2+ channels. Disruption of this equilibrium stimulates the expression of proteins that can lead to cellular transformation, proliferation, invasion and metastasis. One of the regulators of intracellular Ca2+ signaling is SERCA2 [
SERCA2 expression in CRA, CRC and normal mucosal tissues were investigated in the present study. It was observed that SERCA2 expression was variable between normal and neoplastic lesions (CRA and CRC) with significant increase in its expression in neoplastic lesions as none of the normal colonic tissues showed high SERCA2 expression. This was in agreement with previous study which found that the mean expression level of SERCA2 mRNA in malignant colorectal tissues was significantly higher than that in noncancerous tissues [
Furtherly, the SERCA2 expression in CRA neoplastic cells in relation to clinicopathological parameters was studied. It was found that high SERCA2 expression was significantly associated with adenoma with HGD than adenoma with LGD. No significant relation was detected between SERCA2 expression and histological architecture of adenoma.
Besides the fact that the expression of SERCA2 in CRC epithelial cells was tested, it was found that SERCA2 overexpression was significantly related to high tumor grade, advanced TNM staging, presence of LVI and high ITN (but not lymphocytic) infiltrate. These findings were similar to previous studies which showed that colorectal carcinoma with increased SERCA2 expression was significantly associated with serosal invasion, lymph node metastasis and advanced tumor stage compared to those with low SERCA2 expression meaning that SERCA2 may be a crucial biomarker in the development and progression of CRC [
In addition, previous studies reported the key role of intracellular calcium remodeling in colorectal cancer process that includes cellular proliferation, migration, invasion and metastasis [
Our results supported the predominant association of SERCA2 overexpression with poor prognostic factors of CRC, as SERCA2 overexpression in cancer cells is a prerequisite for evasion of apoptosis causing depletion of intracellular Ca2+
Furthermore, inhibition of SERCA2 expression was proved to induce G2/M cycle arrest and growth elimination in SW480 human colon adenocarcinoma epithelial cells
IL-8 is a multifunctional chemokine which was found to induce tumor growth, angiogenesis and metastatic potential in CRC.
IL-8 expression was examined in CRA, CRC as well as in normal colonic mucosa. A significant increase in IL-8 expression rate was detected in CRC more than in CRA and normal colonic mucosa. Similarly, previous studies found that IL-8 is upregulated in CRC compared to CRA and normal colonic mucosa [
Subsequently, IL-8 expression was studied in CRA in relation to clinicopathological parameters, where no significant relations between IL-8 overexpression and CRA type or location were found, while high IL-8 immunostaining was significantly associated with CRA showing HGD. A Similar finding in other study which found that IL-8 mRNA level was significantly elevated with increased degree of dysplasia in the adenomas [
In addition, IL-8 expression in neoplastic cells of CRC was investigated, and it was detected that IL-8 protein overexpression was significantly linked to high tumor grade, presence of LVI, increased tumor size, regional lymph node, distant metastasis and high intra-tumoral inflammatory infiltrate. No significant relation was detected between IL-8 expression and tumor site or histological type.
Parallel to our results, previous studies showed that IL-8 expression is significantly upregulated in CRC neoplastic cells and this upregulation was associated with increased tumor growth, invasion and distant metastasis [
The growth signaling pathway (MAPK) is stimulated by interaction of IL-8 and its receptors that may be expressed in tumor associated stromal cells particularly the inflammatory cells [
It is noteworthy that, in the current study, high expression of TSP50, SERCA2 and IL-8 were significantly associated with high intra-tumoral inflammatory infiltrate in CRC. This finding supports the idea of the role of inflammatory microenvironment in increasing mutation rates and enhancing the division of mutated cells. This may be due to release of reactive oxygen species (ROS) from activated inflammatory cells which are able to promote DNA mutation and genomic instability in the epithelial cells [
Regarding the correlation between the expression of TSP50, SERCA2 and IL-8 in CRC, significant positive correlation was found between the three biomarkers’ expression in neoplastic epithelial cells. This finding could be explained by the fact that both inflammatory process and tumorigenesis are related to disruption of Ca2+ homeostasis which is involved in DNA and gene methylation [
Based on our findings. The aberrant expression of TSP50, SERCA2 and IL-8 in CRA and CRC compared to normal colonic mucosa could represent a different molecular transformation pathway. Also, increased expression of these biomarkers in neoplastic epithelial cells of CRC is associated with adverse prognostic factors through facilitating several hallmarks of cancer and could be considered as independent prognostic factors. Therefore, combination immunotherapy targeting TSP50, SERCA2 and IL-8 molecules would be a valuable effective therapy for inhibiting colorectal carcinoma initiation, proliferation and metastasis.
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by Tanta University Faculty of medicine. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
The authors confirm contribution to the paper as follows: study conception and design HY and MA; analysis and interpretation of results: MA; draft manuscript preparation: MA and DR. All authors reviewed the results and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.