Implications of ABCC4-Mediated cAMP Eflux for CRC Migration

Abstract

Colorectal cancer (CRC) presents significant molecular heterogeneity. The cellular plasticity of epithelial to mesenchymal transition (EMT) is one of the key factors responsible for the heterogeneous nature of metastatic CRC. EMT is an important regulator of ATP binding cassette (ABC) protein expression; these proteins are the active transporters of a broad range of endogenous compounds and anticancer drugs. In our previous studies, we performed a transcriptomic and functional analysis of CRC in the early stages of metastasis induced by the overexpression of Snail, the transcription factor involved in EMT initiation. Interestingly, we found a correlation between the Snail expression and ABCC4 (MRP4) protein upregulation. The relationship between epithelial transition and ABCC4 expression and function in CRC has not been previously defined. In the current study, we propose that the ABCC4 expression changes during EMT and may be differentially regulated in various subpopulations of CRC. We confirmed that ABCC4 upregulation is correlated with the phenotype conversion process in CRC. The analysis of Gene Expression Omnibus (GEO) sets showed that the ABCC4 expression was elevated in CRC patients. The results of a functional study demonstrated that, in CRC, ABCC4 can regulate cell migration in a cyclic nucleotide-dependent manner.

Keywords: ABC transporters; ABCC4 protein; colon cancer; metastasis.

Figure 1

ABCC4 (A), ABCG2 (F), and EMT marker (B–E) mRNA expression in CRC and normal tissue. Microarray data from the public Gene Expression Omnibus (GEO) database (GSE18105, GSE44861, and GSE32323: [20] were analyzed with the respective n for c (cancer) and n (normal): GSE18105 n_c = 57 n_n = 10 (primary tumors only); GSE44861 n_c = 56, n_n = 55; and GSE32323 n_c = 17 n_n = 17. Normality test (Shapiro–Wilk) was performed, followed by the Mann–Whitney U test (*)—* p < 0.05; ** p < 0.005; *** p < 0.001, no statistically significant—no indicator. Additionally, all the normally distributed samples were tested using t-test (#) # p < 0.05; ### p < 0.001. Data density distribution is presented in the Supplementary Materials, Figure S2.

Figure 2

TGFβ pathway and ABCC4 mRNA expression analysis. TGFβ1/2 (A) and TGFβR1/2 (D) expression analysis in CRC patient tissue. Data obtained from the GSE18105 data set, cancer n = 94, normal n = 16. Correlation of ABCC4 expression and TGFβ1 (B) or TGFβ2 (C) or TGFβR1 (E) or TGFβR2 (F) expression, n = 110 [20]. Sample data sets were tested with the Shapiro–Wilk test, presenting a normal distribution, followed by the T-test. Pearson’s correlation coefficient (PCC): 0–0.25 no PCC, 0.25–0.5 low PCC, 0.5–0.75 moderate PCC, 0.75-1 strong PCC. Comparison of Pearson’s and Spearman’s correlation values in Figure S1A, Supplementary Materials. Data density distribution (Figure 2A,D) produced with SinaPlot is presented in Supplementary Materials, Figure S2. * p < 0.05; ** p < 0.005.

Figure 2

TGFβ pathway and ABCC4 mRNA expression analysis. TGFβ1/2 (A) and TGFβR1/2 (D) expression analysis in CRC patient tissue. Data obtained from the GSE18105 data set, cancer n = 94, normal n = 16. Correlation of ABCC4 expression and TGFβ1 (B) or TGFβ2 (C) or TGFβR1 (E) or TGFβR2 (F) expression, n = 110 [20]. Sample data sets were tested with the Shapiro–Wilk test, presenting a normal distribution, followed by the T-test. Pearson’s correlation coefficient (PCC): 0–0.25 no PCC, 0.25–0.5 low PCC, 0.5–0.75 moderate PCC, 0.75-1 strong PCC. Comparison of Pearson’s and Spearman’s correlation values in Figure S1A, Supplementary Materials. Data density distribution (Figure 2A,D) produced with SinaPlot is presented in Supplementary Materials, Figure S2. * p < 0.05; ** p < 0.005.

Figure 3

ABCC4 protein expression level in CRC cell lines. Western blot performed in standard reducing SDS PAGE conditions using goat anti ABCC4 (#PA5 18315, Thermo Scientific) and rabbit anti ABCG2 (#ORB 155559 Biorbyt). (A) Protein expression level of ABCC4 and ABCG2 in HT-29 stably overexpressing transcription factor Snail (HT29/Snail) and control HT-29. ABCC4 level in the membrane fraction (obtained by biotynylation using EZ-Link Sulfo -NHS-Biotin Thermo Scientific kit) of HT-29 control cells and HT-29 Snail n = 3. (B) ABCC4 protein expression level in CRC cells in different states of EMT: CCD841CoN (most epithelial), CaCo-2 (moderate EMT), and Colo-320 (most mesenchymal) n = 3. (C) ABCC4 protein abundance in Extracellular Vesicles (EVs) released from HT-29 control cells and two HT-29 stably overexpressing transcription factor Snail clones (HT-29/Snail and HT-29/Snail17), n = 2. (D) Intracellular cAMP level measurement. Accumulation of cAMP in HT29 cells was measured using a cAMP competitive kit (#581001 Cayman Chemical). Cells were incubated for 24 h with MK571 20 µM, or untreated ones were assayed according to the manufacturer’s protocol. Calculation were conducted using the Cayman data sheet. cAMP concentration of HT29 was set as 100%. T-test performed, n = 5; * p < 0.05; ** p < 0.005; *** p < 0.001. NS—not statistically significant. (E) PKA phosphorylation profile analysis. HT29 Snail cells were seeded on a 6-well plate. Then, 24 h after, full growth medium was changed into starving (FBS free) medium for 24 h. Next, 20uM of MK571 was added to cells for 60, 30, 5, and 1 min. Cells without the starving procedure were used as a positive control, and negative control cells were not treated with MK571. Phosphorylation profile analysis was performed using phospho-(ser/thr) PKA Substrate Antibody #9621 (Cell Signaling Technology). Significant time- (exposure) related impact on the phosphorylation profile was observed for 42 kDa and 95–100 kDa proteins in HT29 Snail cells compared to no time-related changes in control cells, n = 3. (F) HT-29/Snail PKA phosphorylation profile analyzed with densitometry; statistical significance estimated using T-test. * p < 0.05; ** p < 0.005; *** p < 0.001. NS—not statistically significant.

Figure 4

MK571 impact on CRC cells migratory abilities. (A) HT29 cells (control or overexpressing Snail) were grown to confluence on 6 well plate, next, wounded across the cell monolayer. New medium containing 20 μM MK571 was added. Wounded area was visualized after 0, 2, 4, 6 and 24 h—and presented in using Nikon Eclipse TE 2000-U microscope (Nikon, Japan) and calculated by ImageJ software [32]. Cell motility was estimated through the quantification of the % of recovery using the equation: R (%) = [1 − (wound area at Tt/wound area at T0)] × 100,where T0 is the wounded area at 0 h and Tt is the wounded area after t; n = 3; * HT-29/Snail (w/wo MK571) vs. HT-29 control; # HT-29/Snail MK571 vs. HT-29/Snail * p < 0.05; ** p < 0.005; *** p < 0.001, NS—no statistically significant. (B) Representative picture of wound healing assay. (C) Gelatinolysis mediated by HT-29 measured by in-situ zymography. The pericellular proteolytic abilities of HT-29 were analyzed by a measure of the increase in FITC fluorescent intensity from digested DQ gelatine relativized to control cells presented as 100%; n = 3 (D) HT29 cells, transwell assay. Cells incubated for 24 h with MK571 20 µM, or untreated once were seeded on Matrigel coated transwell inserts in the upper chamber in medium supplemented with 0.1% bovine albumin serum (BSA) (w/wo 20 µM MK571). Full medium in lower chamber served as chemoattractant for cell invasion. Membrane were cut out and all cells from membrane were calculated after 6 h of incubation followed by hematoxylin/eosin staining. Number of control HT-29 cells that transmigrate into transwell membrane through 8 µM pores covered with Matrigel was set as 100%, next number of other cells was calculated and presented as % of control. (E) CaCo-2 cells, transwell assay. Cells were incubated for 24 h with MK571 20 µM, or untreated once were seeded on un-coated transwell inserts (8 µM pores) in the upper chamber in 2% BSA medium (w/wo 20 µM MK571). Full medium in lower chamber served as chemoattractant for cell migration. Cells were calculated in randomly assigned areas after 3 h of incubation followed by hematoxylin/eosin staining. Interquartile range (Q1–Q3) is shown as gray box with median (Q2) with all data points from all (n = 3) experiments overlap on the box plot. (F) CaCo-2 cells, wound healing assay. Cells were seeded on collagen coated 6-well plates to full confluence. Next wound was done across cell monolayer, rinsed with phosphate buffer (PBS) PBS. Next fresh medium w/wo 20 µM MK571 was added. Cells were visualized every 2 h and % of wound enclosure was calculated as in A). * p < 0.05; ** p < 0.005; *** p < 0.001, NS—not statistically significant.

Figure 4

MK571 impact on CRC cells migratory abilities. (A) HT29 cells (control or overexpressing Snail) were grown to confluence on 6 well plate, next, wounded across the cell monolayer. New medium containing 20 μM MK571 was added. Wounded area was visualized after 0, 2, 4, 6 and 24 h—and presented in using Nikon Eclipse TE 2000-U microscope (Nikon, Japan) and calculated by ImageJ software [32]. Cell motility was estimated through the quantification of the % of recovery using the equation: R (%) = [1 − (wound area at Tt/wound area at T0)] × 100,where T0 is the wounded area at 0 h and Tt is the wounded area after t; n = 3; * HT-29/Snail (w/wo MK571) vs. HT-29 control; # HT-29/Snail MK571 vs. HT-29/Snail * p < 0.05; ** p < 0.005; *** p < 0.001, NS—no statistically significant. (B) Representative picture of wound healing assay. (C) Gelatinolysis mediated by HT-29 measured by in-situ zymography. The pericellular proteolytic abilities of HT-29 were analyzed by a measure of the increase in FITC fluorescent intensity from digested DQ gelatine relativized to control cells presented as 100%; n = 3 (D) HT29 cells, transwell assay. Cells incubated for 24 h with MK571 20 µM, or untreated once were seeded on Matrigel coated transwell inserts in the upper chamber in medium supplemented with 0.1% bovine albumin serum (BSA) (w/wo 20 µM MK571). Full medium in lower chamber served as chemoattractant for cell invasion. Membrane were cut out and all cells from membrane were calculated after 6 h of incubation followed by hematoxylin/eosin staining. Number of control HT-29 cells that transmigrate into transwell membrane through 8 µM pores covered with Matrigel was set as 100%, next number of other cells was calculated and presented as % of control. (E) CaCo-2 cells, transwell assay. Cells were incubated for 24 h with MK571 20 µM, or untreated once were seeded on un-coated transwell inserts (8 µM pores) in the upper chamber in 2% BSA medium (w/wo 20 µM MK571). Full medium in lower chamber served as chemoattractant for cell migration. Cells were calculated in randomly assigned areas after 3 h of incubation followed by hematoxylin/eosin staining. Interquartile range (Q1–Q3) is shown as gray box with median (Q2) with all data points from all (n = 3) experiments overlap on the box plot. (F) CaCo-2 cells, wound healing assay. Cells were seeded on collagen coated 6-well plates to full confluence. Next wound was done across cell monolayer, rinsed with phosphate buffer (PBS) PBS. Next fresh medium w/wo 20 µM MK571 was added. Cells were visualized every 2 h and % of wound enclosure was calculated as in A). * p < 0.05; ** p < 0.005; *** p < 0.001, NS—not statistically significant.

Figure 5

MK571 increased motility of EndoMT undergoing HMEC-1 cells. ABCC4 level in HMEC-1 and HMEC-1 overexpressing Snail cells (HMEC-1/Snail). (A) HMEC-1 were grown to confluence on 6-well plate, and transiently transfected with pcDNA/Snail and wounded across monolayer as described in [31]. New medium containing MK571 was added. (B) Representative image of HMEC-1 control or HMEC-1/Snail cells in wound healing assay. (C) Wounded area was visualized after 0, 2, 4 and 6 h using Nikon Eclipse TE 2000-U microscope (Nikon, Japan) and calculated by ImageJ software [32]. Cell motility was estimated through the quantification of the % of recovery using the equation: R(%) = [1 − (wound area at Tt/wound area at T0)] × 100,where T0 is the wounded area at 0 h and Tt is the wounded area after 2 or 4 h. * p < 0.05; ** p < 0.005; n = 3. (D) HMEC-1 treated w/wo TGF-β receptor inhibitor were grown to confluence on 6 well plate, incubated for 48 h with 10ng/mL TGF-β2 in starving condition as described in [31 and wounded. Wounded area was visualized and analyzed as in (C).

Figure 6

Irinotecan affects CRC migration. HT29 control and HT29/Snail cells were seeded on 24 well plate to confluence for 24 h. Next, wound was done across monolayer and fresh medium was added w/wo 2.5 µM irinotecan (final concentration). Wounded area was visualized after every 2 h by Spark multimode microplate reader (TECAN, Swizerland). Wounded area was calculated by ImageJ software [32]. Cell motility was estimated through the quantification of the % of recovery using the equation: R(%) = [1 − (wound area at Tt/wound area at T0)] × 100,where T0 is the wounded area at 0 h and Tt is the wounded area after 2 or 4 h. * p < 0.05; ** p < 0.005, NS—no statistically significant (all HT29 control vs HT29 control + 2.5 µM irinotecan were considered NS), n = 3.