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This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (
Colorectal cancer (CRC) is the third most common malignancy and the fourth in cancer related deaths worldwide [
Activins are plethoric proteins that belong to the transforming growth factor (TGF)-β family and normally mediate their cellular actions via Smad4 [
Act-A is the most studied isoform in CRC and the findings of several studies have demonstrated paradoxical actions for the protein, a phenomenon known as the “TGF-β paradox” or “molecular switch” [
The available reports regarding the roles of Act-B, Act-AB, and FS in colon carcinogenesis are scarce [
Ethical approval was granted from the Institutional Review Boards of King Faisal Specialist Hospital and Research Centre (KFSH&RC; #RC-J/448/39) in Jeddah and King Abdullah Medical City (KAMC; #19-498) in Makkah prior to recruitment. Paired malignant and normal specimens were collected from two sources, namely archived Formalin-Fixed Paraffin-Embedded (FFPE), alongside another set of fresh tumours with their corresponding adjacent normal tissues following surgical resection. All patients from the FFPE and fresh tissue cohorts were Saudi citizens diagnosed with primary sporadic CRC and did not receive neoadjuvant chemo/radiotherapy before their curative/palliative surgery. Cases with inherited or recurrent CRC were excluded. The FFPE cohort of malignant, and their corresponding normal tissues, were obtained from 90 patients who had their surgery between January 2016 and June 2018. The tissue blocks were retrieved from the archives of the histopathology departments of both study sites following examination by expert histopathologists to assure adequacy.
The fresh specimens were collected from 40 patients between July 2018 and March 2020. Excised colon tissue was immediately transferred to the histopathology department and intratumoral, together with adjacent non-cancerous tissue specimens located at least 10 cm away from the tumour margins, were cut by an expert histopathologist after gross examination. The specimens were instantly submerged in RNA
The final diagnosis and the histopathological staging were conducted by a consultant histopathologist according to the criteria of the 8th edition of the American Joint Committee on Cancer tumour-node-metastasis (TNM) staging system. As per the pathology and surgical reports, FFPE and fresh tumours located from the cecum to the proximal margin of splenic flexure were classified as RSC, whereas tumours located from the splenic flexure to the rectum were considered left-sided cancer (LSC).
All the primary antibodies were from Thermo Fisher Scientific. While rabbit polyclonal IgG antibodies were used to localise Smad4 (#PA5-34806), βB-subunit (#PA5-50818) and FS (#PA5-114319), the βA-subunit was detected by goat polyclonal IgG antibodies (#PA5-47004). Tissue sections (5 μm) were dewaxed in xylene, emersed in graded ethanol series, and then washed with deionized water. An avidin-biotin horseradish peroxidase technique was applied, and endogenous peroxidases were blocked with BLOXALL® Endogenous Blocking Solution (#SP-6000-100; Vector Laboratories Inc., CA, United States). The slides were then washed in PBS for 10 min and normal goat (for rabbit 1ry antibodies), or rabbit (for goat 1ry antibodies) serum provided with the VECTASTAIN® rabbit (#PK-6101) or goat (#PK-6105) Elite ABC-HRP Kits (Vector Laboratories Inc.) were then added to the sections for 30 min. The primary antibodies were subsequently added (1:150 concentration for all) followed by overnight incubation at 4°C. In the next day, the provided rabbit anti-goat or goat anti-rabbit biotinylated secondary antibodies alongside the avidin/biotin complex in the VECTASTAIN® rabbit or goat Elite ABC-HRP Kits were prepared and used as per the manufacturer’s instructions. The negative control sections were treated similar to all other slides, but the primary antibodies were replaced with their equivalent isotype rabbit (#sc-2027) or goat (#sc-2028) IgG antibodies (Santa-Cruz Biotechnology; CA, United States). The sections were observed on a Leica DMi8 microscope (Leica Microsystems, Wetzlar, Germany) and Red/Green/Blue (RGB) digital images were captured from 10 non-overlapping fields with a ×20 objective.
The protein expression in the captured images was digitally analysed by ImageJ software (
Fresh normal and neoplastic colonic tissues weighing 0.5 g each were placed in 1 ml RIPA lysis buffer (#89900) with protease inhibitors (#78429; Thermo Fisher Scientific). Following centrifugation, the total protein concentrations were measured in the supernatants by a BCA Protein Assay (#A53225; Thermo Fisher Scientific). The protein samples were diluted with ultrapure deionized water for the final concentrations of 500 µg/ml. Act-A (#SEA001Hu), Act-B (#SEA170Hu), Act-AB (#SEA158Hu), and FS (#SEA391Hu) concentrations were then measured by ELISA using human specific kits (Cloud-Clone Corp.; TX, United States). All samples were processed in duplicate on a fully automated ELISA system (Human Diagnostics; Wiesbaden, Germany). The tissue concentrations of each protein were compared between the normal and cancerous tissues of each patient as well as between the cancer TNM stages (I/II vs. III/IV), tumour anatomical locations (RSC vs. LSC), and Smad4 status (N-S4 vs. L-S4).
All reagents were from Thermo Fisher Scientific. Total RNA was extracted by the PureLink™ RNA Mini Kit (#12183025) for fresh tissues and PureLink™ FFPE RNA Isolation Kit (#K156002) for archived specimens. The RNA quality and quantities were assessed by Qubit4 Fluorometer (Thermo Fisher Scientific).
The cDNA was synthesised from 1 μg of total RNA by a SuperScript™ VILO™ cDNA Synthesis Kit (#11754250). Biological replicates PCR reactions were performed in triplicate wells on an ABI® 7500 system using the power SYBR Green master mix. Each well had 10 µl SYBR Green, 7 µl DNase/RNase free water, 1 µl of each primer (5 pmol;
The Smad4-intact SW480 [
For cell cycle analysis, 2 × 105 SW480 and 3 × 105 HT29 cells were seeded in 6-well plates for 24 h, whereas for immunofluorescence, cells were seeded in 8-well chamber slides (#229168; Celltreat™ Scientific Products; MA, United States) at 8,000 (SW480) and 16,000 (HT29) cells/well, also for 24 h. Concentrations (IC50) of Act-A (100 ng/ml), Act-AB (200 ng/ml) and FS (100 ng/ml) were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) cytotoxicity assay (data not shown). Act-B had little effect at the highest tested dose (500 ng/ml), and was therefore used at 200 ng/ml, matching the highest concentration of the activins, described above.
Following treatment with activins or follistatin, the cells were processed for cell cycle analysis according to Aslam et al. [
Following activins or follistatin treatments for 24 h, the cells in the 8-well chamber slides were washed and then fixed for 15 min with 4% paraformaldehyde (#sc-281692; Santa-Cruz Biotechnology Inc.). The cells were then washed, permeabilised with 0.25% Triton X100 (#T8787; Sigma-Aldrich Co.; MO, United States) for 20 min, washed twice with PBS, and blocked for 30 min with normal donkey serum (#sc-2044; Santa-Cruz Biotechnology Inc.). Untreated SW480 and HT29 cells were also processed to confirm Smad4 status using 1:150 concentration of polyclonal rabbit IgG antibodies (#PA5-34806; Thermo Fisher).
The co-expression of cyclin D1 (CCND1) with cyclin-dependent kinase inhibitor-1B (p27), cyclin D3 (CCND3) with cyclin-dependent kinase inhibitor-1A (p21), survivin with caspase-8 (Casp-8), and B-cell lymphoma 2 (BCL2) with cleaved Casp-3 was examined in duplicate wells. The primary antibodies against p21 (#2947), p27 (#3686), survivin (#2808) and cleaved casp-3 (#9661) were monoclonal rabbit IgG antibodies (Cell Signaling Technology Inc., MA, United States), whereas CCND1 (#sc-8396), CCND3 (#sc-6283), BCL2 (#sc-7382) and Casp-8 (#sc-56070) were mouse monoclonal IgG antibodies (Santa-Cruz Biotechnology Inc.). Each set of primary mouse and rabbit antibodies was added to two wells/slide (1:200 concentration for all antibodies) and the slides were incubated for 3 h at room temperature. Following washing, the cells were incubated for 60 min with a mixture of tagged highly cross-adsorbed secondary donkey anti-rabbit (#A-31572; Alexa Fluor 555) and anti-mouse (#A-21202; Alexa Fluor 488) IgG antibodies (Thermo Fisher Scientific). The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; #D3571; Thermo Fisher Scientific), the detachable plastic wells were then removed, and the slides were cover-slipped with a permanent fluorescence mounting medium (#S3023; Dako, CA, United States).
All wells were observed with a Leica DMi8 microscope and digital images were captured within the same session from 10 random non-overlapping fields/well using a ×40 objective. The IF staining intensities of each targeted protein were measured by digital image analysis using the ImageJ software and are expressed as arbitrary units/cell numbers in analysed images, as previously described [
All variables were assessed for normality by the Kolmogorov and Smirnov’s test and homogeneity by Levene test using SPSS version 25. Ordinal and discontinuous data are presented as numbers and percentages, and cross-tabulation followed by Chi-square (χ2) test were used for frequency analysis. Independent Student’s t or Mann-Whitney U tests were used to compare between two groups based on normality. Continuous data are shown either as mean ± standard deviation (SD) or median with interquartile range (IQR; 25th–75th percentiles), depending on data normality. Correlations were determined by Pearson’s or Spearman’s tests according to data normality.
The FFPE (
The clinicopathological characteristics according to sample source.
Fresh samples ( |
FFPE samples ( |
|
|
---|---|---|---|
Mean ± SD of Age (year) | 62.1 ± 11.9 | 62.8 ± 12.2 | 0.8 |
Gender | |||
Male | 19 (14.6%) | 50 (38.4%) | 0.4 |
Female | 21 (16.2%) | 40 (30.8%) | |
Tumour sidedness | |||
Right-sided | 13 (10%) | 26 (20%) | 0.4 |
Left-sided | 27 (20.8%) | 64 (49.2%) | |
Tumour infiltration (T stage) | |||
T1 | 2 (1.6%) | 4 (3.1%) | 0.9 |
T2 | 7 (5.4%) | 14 (10.8%) | |
T3 | 19 (14.6%) | 45 (34.6%) | |
T4 | 12 (9.2%) | 27 (20.7%) | |
Median (IQR) of tumour volume (cm3) | 21.2 (9.6–49.5) | 18 (8.9–39.1) | 0.08 |
Regional lymph node (N stage) | |||
N0 | 18 (13.9%) | 55 (42.3%) | 0.1 |
N1 | 15 (11.5%) | 19 (14.6%) | |
N2 | 7 (5.4%) | 16 (12.3%) | |
Distant metastasis (M stage) | |||
M0 | 35 (26.9%) | 77 (59.2%) | 0.7 |
M1a | 5 (3.9%) | 13 (10%) | |
Histology | |||
Adenocarcinoma | 35 (26.9%) | 73 (56.1%) | 0.4 |
Mucinous | 5 (3.9%) | 17 (13.1%) | |
Differentiation | |||
Poor | 4 (3.1%) | 11 (8.4%) | 0.9 |
Moderate | 28 (21.5%) | 63 (48.5%) | |
Well | 8 (6.2%) | 16 (12.3%) | |
AJCC TNM stages | |||
Stages I/II (early) | 15 (11.5%) | 45 (34.6%) | 0.2 |
Stages III/IV (advanced) | 25 (19.3%) | 45 (34.6%) |
Right-sided tumours were less prevalent (
The clinicopathological characteristics according to tumour side. Bold values indicate statistical significance.
Right-sided tumours ( |
Left-sided tumours ( |
|
|
---|---|---|---|
Sample source | |||
Archived samples (FFPE) | 26 (20%) | 64 (49.2%) | 0.7 |
Fresh samples | 13 (10%) | 27 (20.8%) | |
Mean ± SD of Age (year) | 64.1 ± 13.1 | 61.6 ± 11.4 | 0.3 |
Gender | |||
Male | 23 (17.7%) | 46 (35.4%) | 0.4 |
Female | 16 (12.3%) | 45 (34.6%) | |
Tumour infiltration (T stage) | |||
T1 | 0 (0%) | 6 (4.6%) |
|
T2 | 0 (0%) | 21 (16.1%) | |
T3 | 20 (15.4%) | 44 (33.9%) | |
T4 | 19 (14.6%) | 20 (15.4%) | |
Median (IQR) of tumour volume (cm3) | 29 (15–54) | 12 (7–25) |
|
Regional lymph node (N stage) | |||
N0 | 24 (18.4%) | 49 (37.7%) | 0.4 |
N1 | 7 (5.4%) | 27 (20.8%) | |
N2 | 8 (6.2%) | 15 (11.5%) | |
Distant metastasis (M stage) | |||
M0 | 25 (19.3%) | 87 (66.9%) |
|
M1a | 14 (10.7%) | 4 (3.1%) | |
Histology | |||
Adenocarcinoma | 26 (20%) | 82 (63.1%) |
|
Mucinous | 13 (10%) | 9 (6.9%) | |
Differentiation | |||
Poor | 11 (8.5%) | 4 (3.1%) |
|
Moderate | 27 (20.8%) | 64 (49.2%) | |
Well | 1 (0.7%) | 23 (17.7%) | |
AJCC TNM stages | |||
Stages I/II (early) | 13 (10%) | 47 (36.1%) | 0.06 |
Stages III/IV (advanced) | 26 (20%) | 44 (33.9%) |
The Smad4 mRNA was markedly decreased in the early-stage (3.2-fold) and advanced-stage (11.1-fold) neoplastic tissues relative to their corresponding normal specimens (
Smad4 protein was exclusively localised in the epithelial nuclei and the expression in normal tissues was comparable between the FFPE and fresh samples, anatomical sites, and cancer stages (
Relative expression (median) of Smad4 gene and protein in addition to the protein localisation by IHC in the FFPE (
The overall median of the Smad4 IHC scores in the FFPE and fresh malignant sites was 130.4 (96.2–157.64), and 26 patients (20%) showed IHC scores ≤ the 20th percentile cut-off value (87.44) and were classified as the L-S4 group. Moreover, the frequencies of low Smad4 protein were not significantly different between the FFPE and fresh malignant specimens, and the age was comparable between the N-S4 and L-S4 groups (
The clinicopathological characteristics according to Smad4 status in the FFPE and fresh malignant tissues (
Normal Smad4 ( |
Low Smad4 ( |
|
|
---|---|---|---|
Sample source | |||
Archived samples (FFPE) | 71 (54.6%) | 19 (14.6%) | 0.6 |
Fresh samples | 33 (25.4%) | 7 (5.4%) | |
Mean ± SD of Age (year) | 62.3 ± 11.9 | 62.7 ± 12.1 | 0.8 |
Gender | |||
Male | 58 (46.6%) | 11 (8.5%) | 0.2 |
Female | 46 (35.4%) | 15 (11.5%) | |
Tumour anatomical site | |||
Right-sided tumour | 22 (16.9%) | 17 (13.1%) |
|
Left-sided tumour | 82 (63.1%) | 9 (6.9%) | |
Tumour infiltration (T stage) | |||
T1 | 5 (3.8%) | 1 (0.7%) |
|
T2 | 21 (16.2%) | 0 (0%) | |
T3 | 53 (40.8%) | 11 (8.5%) | |
T4 | 25 (19.2%) | 14 (10.8%) | |
Median (IQR) of tumour volume (cm3) | 12.25 (8.5–25) | 38.5 (19.5–55.75) |
|
Regional lymph node (N stage) | |||
N0 | 60 (46.2%) | 13 (10%) |
|
N1 | 32 (24.6%) | 2 (1.5%) | |
N2 | 12 (9.2%) | 11 (8.5%) | |
Distant metastasis (M stage) | |||
M0 | 101 (77.7%) | 11 (8.5%) |
|
M1a | 3 (2.3%) | 15 (11.5%) | |
Histology | |||
Adenocarcinoma | 93 (71.5%) | 15 (11.5%) |
|
Mucinous | 11 (8.5%) | 11 (8.5%) | |
Differentiation | |||
Poor | 7 (5.4%) | 8 (6.2%) |
|
Moderate | 75 (57.7%) | 16 (12.3%) | |
Well | 22 (16.9%) | 2 (1.5%) | |
AJCC TNM stages | |||
Stages I/II (early) | 59 (45.4%) | 1 (0.7%) |
|
Stages III/IV (advanced) | 45 (34.6%) | 25 (19.3%) |
The mRNA levels of the targeted genes in normal colonic tissues, from the FFPE and fresh cohorts of specimens, were similar between the RSC and LSC as well as between the early and late malignancy stages within each set of specimens (
Relative expression (median) of activin β-subunits and follistatin mRNAs in the FFPE (
The β-subunits and FS mRNAs were also equal between the normal specimens corresponding to the N-S4 and L-S4 cancerous tissues. However, the βA-subunit (34.9; IQR: 13.52–39.13) and FS (12.57; IQR: 8.35–14.88) mRNAs were markedly higher in the L-S4 malignant specimens relative to the N-S4 tumours (9.85; IQR: 7.64–14.35 and 0.78; IQR: 0.48–8.85, respectively;
The antibodies against the β-subunits and FS clearly labelled the cytoplasm of normal luminal and glandular colonic epithelia (
Localisation of activin βA- and βB-subunits alongside follistatin proteins by immunohistochemistry (IHC) in the FFPE (
In cancerous tissues, the βA-subunit was mainly localised in the cytoplasm of carcinomas with scarce stromal staining, and the early and late cancer stages showed markedly (1.4-fold and 2.3-fold, respectively) higher expression relative to their corresponding normal tissues (
Moreover, the βB-subunit protein was equal in the normal specimens from the N-S4 (188.2; IQR: 166.8–210.8) and L-S4 (176.8; IQR: 159.8–187.6) groups. However, the βA-subunit (209.7; IQR: 185.9–237.1 vs. 219.8; IQR: 202.7–253, respectively;
The Act-A, Act-AB, Act-B, and FS concentrations were similar between the right and left-sided normal specimens as well as between normal tissues obtained from the early and late stages of CRC (
Concentrations (mean ± SD) of activin-A, activin-B, activin-AB and follistatin in the fresh cohort of paired malignant and normal colonic tissues (
The normal tissues from the N-S4 and L-S4 had equal levels of Act-A (299.3; IQR: 260.7–333.1 vs. 291; IQR: 255.6–321 pg/ml), Act-AB (268.6; IQR: 187.5–304.5 vs. 268.2; IQR: 218.1–287.1 pg/ml), Act-B (304.6; IQR: 274–363.1 vs. 328.4; IQR: 277–363.1 pg/ml), and FS (4.2; IQR: 3.8–5.6 vs. 4; IQR: 3.6–4.8 ng/ml). In contrast, the L-S4 malignant tissues had markedly higher Act-A (1332.5; IQR: 791–1638.8 vs. 649.2; IQR: 535.9–794 pg/ml;
Act-A and FS intratumoral levels correlated together directly (r = 0.722;
Spearman’s Correlations between the clinicopathological characteristics of fresh colorectal cancer samples (
Activin-A | Activin-B | Activin-AB | Follistatin | |
---|---|---|---|---|
Age | 0.119 | 0.024 | 0.072 | −0.056 |
Gender | 0.232 | −0.071 | −0.310 | 0.176 |
Right-sided cancer | 0.321* | -0.011 | 0.243 | 0.331* |
T stage | 0.397* | 0.229 | −0.316* | 0.515** |
Tumour Size | 0.645** | 0.240 | −0.564*** | 0.777*** |
N Stage | 0.498** | 0.217 | −0.641*** | 0.708*** |
Numbers of positive lymph nodes | 0.500** | 0.216 | −0.618*** | 0.727*** |
M Stage | 0.534*** | -0.380* | −0.409* | 0.311 |
Mucinous Carcinoma | 0.351* | 0.131 | −0.154 | 0.475** |
Poor differentiation | 0.425** | −0.270 | −0.391* | 0.369* |
Low Smad4 expression | −0.499** | 0.091 | 0.408** | −0.430** |
*
**
***
Smad4 protein was detected in the untreated SW480 cells and showed perinuclear localisation, whereas it was negligible in the HT29 cells (
Percentages of cells (mean ± SD) in the different phases of cell cycle in untreated control cells, and following treatments with Act-A, Act-AB, Act-B or FS for 24 h in the
On the other hand, Act-A significantly reduced the numbers of HT29 cells in the sub-G1 phase, whilst simultaneously increasing the percentages in the G0/G1 and S-phases, relative to control cells (
In the SW480 cells, CCND1 (
Co-expression of CCND1 (green) with p27 (red) proteins by immunofluorescence in untreated control cells and following 24 h treatment with Act-A (100 ng/ml), Act-AB (200 ng/ml), Act-B (200 ng/ml) or FS (100 ng/ml) in the SW480 and HT29 cell lines (Scale bar = 8 μm). In addition, the arbitrary scores of the immunofluorescent stain intensity/cell (mean ± SD) for each protein are shown as graph bars (a =
Co-expression of CCND3 (green) with p21 (red) proteins by immunofluorescence in untreated control cells and following 24 h treatment with Act-A (100 ng/ml), Act-AB (200 ng/ml), Act-B (200 ng/ml) or FS (100 ng/ml) in the SW480 and HT29 cell lines (Scale bar = 8 μm). In addition, the arbitrary scores of the immunofluorescent stain intensity/cell (mean ± SD) for each protein are shown as graph bars (a =
Interestingly, Act-B only diminished CCND1 in the SW480 cells (
The expression of survivin decreased, whereas Casp-8 increased, significantly in the SW480 cells with Act-A and Act-AB treatments compared with untreated cells (
Co-expression of survivin (red) with Casp-8 (green) proteins by immunofluorescence in untreated control cells and following 24 h treatment with Act-A (100 ng/ml), Act-AB (200 ng/ml), Act-B (200 ng/ml) or FS (100 ng/ml) in the SW480 and HT29 cell lines (Scale bar = 8 μm). In addition, the arbitrary scores of the immunofluorescent stain intensity/cell (mean ± SD) for each protein are shown as graph bars (a =
Co-expression of BCL2 (green) with cleaved Casp-3 (red) proteins by immunofluorescence in untreated control cells and following 24 h treatment with Act-A (100 ng/ml), Act-AB (200 ng/ml), Act-B (200 ng/ml) or FS (100 ng/ml) in the SW480 and HT29 cell lines (Scale bar = 8 μm). In addition, the arbitrary scores of the immunofluorescent stain intensity/cell (mean ± SD) for each protein are shown as graph bars (a =
Although Act-B in the SW480 cells only showed limited increases in Casp-8 protein relative to control, the expression of BCL2, survivin and Casp-3 were equal between both groups (
This study measured activins and follistatin in malignant colon tissues and the results were compared between the early and late clinical stages of cancer according to tumour sidedness and Smad4 expression. Additionally, the effects of activins and follistatin proteins related to cell cycle and expression of apoptosis markers were investigated in the Smad-4 intact SW480 and Smad-mutated HT29 colon cancer cells. Similar to earlier reports from Saudi Arabia [
Our results also revealed progressive increases in the βA-subunit gene and protein together with Act-A concentrations in cancerous specimens relative to their corresponding normal samples, and the late-stage neoplastic tissues had markedly higher amounts than the early stages. Act-A levels in malignant samples also correlated directly with tumour sizes, TNM stages, counts of positive lymph nodes and distant metastasis. The RSC advanced stages and the L-S4 malignant tissues had substantially greater amounts of βA-subunit and Act-A. Moreover, Act-A treatment markedly boosted the numbers of the SW480 cells in sub-G1 and G0/G1-phases, increased the cell cycle inhibitory (p21/p27) and pro-apoptotic (Casp-8/Casp-3) proteins, and decreased the markers of cell cycle progression (CCND1/CCND3) and cell survival (BCL2/survivin). In contrast, Act-A significantly reduced the numbers of HT29 cells in the sub-G1 phase with concurrent increases in the G0/G1 and S phases. While Act-A modestly induced the p21 and p27 proteins in the HT29 cells, it had no effect on the cell proliferation and apoptosis markers.
Correlating with our data, Act-A triggered p21-dependent cell cycle arrest and apoptosis in Smad4-intact colon cancer cells, whereas the deletion of
Little is currently known about the roles of Act-AB and Act-B in CRC biology [
Herein, the βB-subunit mRNA and protein alongside Act-B and Act-AB levels decreased in malignant compared with normal tissues. Moreover, the late-stage cancerous samples showed significantly lower βB-subunit mRNA and protein alongside Act-AB amounts, but not Act-B, compared with early-stage cancers. Act-AB also correlated inversely with Act-A and the tumour characteristics. Although the RSC and LSC neoplasms had equal amounts of Act-AB and Act-B during the early cancer stages, the former group had markedly lower Act-AB in the advanced stages. The Act-AB levels in the L-S4 malignant tissues were also substantially inferior to the N-S4 samples. Additionally, Act-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases, whilst Act-B mainly induced G2/M-phase arrest in the HT29 cells. Moreover, the p21, p27, Casp-8 and Casp-3 proteins increased with concomitant declines in CCND1, CCND3, BCL2 and survivin in both cell lines with Act-AB. This study agrees with many prior reports and, altogether, suggest that Act-AB could be a Smad4-independent tumour suppressor protein in CRC [
FS tightly controls the biological actions of activins by irreversible binding [
This study showed stage-dependent expression of FS gene and protein in malignant tissues that was depicted by initial decreases during the early stages followed by a drastic upsurge in the late stages of cancer. Additionally, the cancerous tissues FS levels showed direct links with Act-A and the TNM categories, whilst correlated negatively with Act-AB. While FS treatment showed no effect on cell cycle in the SW480 cells, it provoked G1-arrest and enhanced the expression of p21, p27 and Casp-8 alongside decreased CCND1, CCND3 and survivin proteins in the HT29 cells. Our findings correlate with previous reports and suggest that the observed aberrant alterations in activins during colon carcinogenesis could be related to an atypical disequilibrium with FS [
In conclusion, activins and follistatin are pathologically altered in CRC and their deregulations were more pronounced in advanced right-sided tumours and with the loss of Smad4 protein, suggesting their important contribution to cancer development and aggressiveness. Moreover, our data suggest that the paradoxical actions of Act-A in CRC could be dependent on Smad4 protein expression, which is commonly lost during the late stages of malignancy. This study is also the first to report consistent decreases in Act-AB with CRC progression and the protein induced cell cycle arrest and increased the markers of apoptosis in the SW480 and HT29 cells, implying that it could act as a Smad4-independent tumour suppressor. On the other hand, FS showed biphasic stage-dependent expression, which might induce the observed abnormalities in colonic activins and subsequently cancer progression. However, further studies are still required to explore the complex connections between activins and their related proteins as well as to investigate their expression and actions in relation to tumour microsatellite status and consensus molecular subtypes to precisely explicate their roles in human CRC.
The original contributions presented in the study are included in the article/
The studies involving human participants were reviewed and approved by the Institutional Review Boards of King Faisal Specialist Hospital and Research Centre (KFSH&RC; #RC-J/448/39; Jeddah, Saudi Arabia) and King Abdullah Medical City (KAMC; #19-498; Makkah, Saudi Arabia) prior to recruitment. The patients/participants provided their written informed consent to participate in this study.
Conceptualization: BR and JZ; Recruitment and Sample collection: MB, AM, SS, HA, HS, SE, TH, and MN; Methodology: JA, SI, MA, MB, and BR; Formal analysis: BR; Investigation: AIA, AKA, MA, and BR; Funding acquisition: BR, AIA, AKA, and JZ; Resources and project administration: BR; Supervision: JZ, SE, AIA, AKA, and BR; Writing—original draft: BR. All authors have read and approved the manuscript.
This project was funded by the Deanship of Scientific Research at Umm Al-Qura University; Project (17-MED-1-01-0068).
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.
The Supplementary Material for this article can be found online at:
Localisation of activin βA- and βB-subunits, follistatin, and Smad4 proteins by immunohistochemistry (IHC) in the FFPE (