Glucose impairs tamoxifen responsiveness modulating connective tissue growth factor in breast cancer cells

Abstract

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER⁺) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

Keywords: adipose tissue; breast cancer; connective tissue growth factor; glucose; tamoxifen.

Figure 1. Effect of glucose on MCF7 cell responsiveness to tamoxifen

(A) MCF7 cells grown in high glucose (25mM; HG) or in low glucose (5.5mM; LG), were treated with estradiol (100nM; E₂) and raising concentration (0.1μM, 1μM, 5μM) of tamoxifen (tam); (B) LG cells were shifted to high glucose (LG→HG) during the treatment with E₂ and 0.1μM tam; (C) HG cells were shifted in low glucose (HG→LG) when treated with E₂and 5μM tam. For all the panels (A), (B) and (C), cell viability was assessed, after four days, by sulforhodamine B assay (see Methods). The results were reported as percentage of viable cells compared to positive control (cells treated with E₂ alone), considered as maximum viability (100%). Data represent the mean ± SD of at least three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^**p<0.01); ^§ denote statistically significant values compared with tam treatment in LG cells (^§p<0.01). ^# denote statistically significant values compared with tam treatment in HG cells (^#p<0.05). See also Supplementary Figure 1.

Figure 2. Effect of glucose on MCF7 cell transcriptome

(A) Standard MA plot (M, log ratios and A, mean average) of normalized (upper quartile) RNA-Seq data for each RefSeq gene. The y-axis reports logFC (fold-change), the x-axis the average log intensity (A) for each gene (indicated as dots). Red dots indicate genes that differentially expressed (P≥0.8) in the HG vs HG->LG comparison. (B) Bar graph showing the most significant gene pathways enriched with DEGs after the shift of MCF7 cells from high glucose (25mM; HG) to low glucose (5.5mM; HG→LG). (C) Heatmap of log-transformed normalized gene expression values for a small subset of highly significant deregulated genes belonging to “Cell cycle” pathway. (D) MCF7 cells grown HG (dotted line) were shifted in LG (HG→LG) during the treatment with estradiol (100nM; E₂). After four days, mRNA expression levels of “Cell cycle” DEGs were determined by qRealTime-PCR (see Methods and Table 1). Data were normalized on Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the mRNA levels of DEGs in HG→LG cells relative to those in HG cells. ^* denote statistically significant values (^*p<0.05, ^**p<0.01, ^***p<0.001). See also Supplementary Figure 1.

Figure 3. Role of glucose-induced CTGF on HG cell responsiveness to tamoxifen

(A) MCF7 cells cultured in high glucose medium (25mM; HG) were transfected (see Methods) with three different siRNAs recognizing CTGF (10nM; CTGF_siRNA) or with a control siRNA (10nM; CTRL_siRNA); (C) HG cells were transfected with three different siRNAs recognizing CYR61 (1nM; CYR61_siRNA cells) or with a control siRNA (1nM; CTRL_siRNA). For both panels (A) and (C), after 6 hours, the cells were feed with complete medium and incubated for further 24 or 48 hours. The efficiency of siRNAs activity was assessed by qRealTime-PCR, as described in Methods. Data were normalized on Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the mRNA expression levels of (A) CTGF in CTGF_siRNA cells and (C) CYR61 in CYR61_siRNA cells, both relative to those in CTRL_siRNA cells (dotted line), after 24 (dark grey columns) and 48 hours (light grey columns) transfection. ^* denote statistically significant values (^**p<0.01; ^***p<0.001). (B) HG cells transfected with selected CTGF siRNA (CTGF_{siRNA 2}) or with control siRNA (CTRL_siRNA) were treated with E₂ (100nM) and tam (5μM); (D) HG cells transfected with selected CYR61 siRNA (CYR61_{siRNA 3}) and with control siRNA (CTRL_siRNA) were treated with E₂ and tam (5μM). For both panels (B) and (D), as positive control, the cells were treated with E₂ alone. After 24 hours, cell viability was determined, by sulforhodamine B assay (see Methods). The results were reported as percentage of viable cells compared with positive control considered as 100% viability. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^*p<0.05, ^**p<0.01, ^***p<0.001); # denote statistically significant values compared with tam treatment in CTRL_siRNA cells (^#p<0.01).

Figure 4. Role of glucose-induced CTGF on LG cell responsiveness to tamoxifen

(A) MCF7 cells grown in low glucose medium (5.5mM; LG) were shifted in high glucose medium (LG→HG) during the treatment with E₂ (100nM). After four days, mRNA expression levels of CTGF were determined by qRealTime-PCR (see Methods). Data were normalized on HPRT gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the mRNA levels of CTGF in LG→HG cells relative to those in LG cells. ^* denote statistically significant values (^**p<0.01). (B) CTGF protein levels in MCF7 chronically exposed to high (HG 25mM - dotted line) or low (LG 5.5mM - solid line) glucose concentrations. One representative experiment is shown. (C) LG cells were treated with E₂ and tam (5μM) in presence or absence of human recombinant CTGF protein at increasing doses (50 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL; rCTGF). As positive control, the cells were treated with E₂ alone. After 4 days, cell viability was assessed by sulforhodamine B assay, as described in Methods. The results were reported as percentage of viable cells compared with positive control, considered as 100% viable cells. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^**p<0.01;^***p<0.001); ^# denote statistically significant values compared with tam treatment in untreated cells (^#p<0.05). See also Supplementary Figure 1. (D) LG cells transfected with selected CTGF siRNA (CTGF_{siRNA 2}) or with control siRNA (CTRL_siRNA) were treated with E₂ (100nM) and tam (0.1μM and 5μM). As positive control, the cells were treated with E₂ alone. After 24 hours, cell viability was determined, by sulforhodamine B assay (see Methods). The results were reported as percentage of viable cells compared with positive control considered as 100% viability. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^*p<0.05, ^**p<0.01, ^***p<0.001); ^# denote statistically significant values compared with tam treatment in CTRL_siRNA cells (^#p<0.01).

Figure 5. Role of CTGF in glucose-induced adipocyte-effect on MCF7 responsiveness to tamoxifen

(A) Mature adipocytes, cultured in 15mM glucose, were incubated in serum free high glucose medium (25 mM; HG). After 8 hours, media were collected - from adipocytes pre-incubated or not in HG (hAdipo-CM and HG hAdipo-CM, respectively) - and applied onto MCF7 cells. As control, the cells were incubated in serum-free medium (15mM and 25mM, respectively). After four days, CTGF mRNA expression levels were determined by qRealTime-PCR, as described in Methods. Data were normalized on Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the expression levels of CTGF in cells treated with conditioned media relative to those in untreated cells (dotted line). ^* denote statistically significant values compared with positive control (^**p<0.01); ^# denote statistically significant values compared with cells treated with hAdipo-CM (^#p<0.05). (B) MCF7 cells were treated with estradiol (100nM; E₂) and tamoxifen (5μM; tam) in presence of HG hAdipo-CM or in absence of it (in serum-free HG medium). As positive control, the cells were treated with E₂ alone. After 4 days, cell viability was determined by sulforhodamine B assay, as described in Methods. (C) MCF7 cells were seeded in the upper chamber of a transwell system with or without mature adipocytes in the lower chamber. MCF7 cells were treated with E₂ and tam (5μM) in serum free HG medium. As positive control, the cells were treated with E₂ alone. After 24 hours, cell viability was assessed by crystal violet, as described in Methods. (D) MCF7 cells transfected (see Methods) with selected siRNAs recognizing CTGF (10nM; CTGF_{siRNA 2}) or with a control siRNA (10nM; CTRL_siRNA) were treated with E₂ (100nM) and tam (5μM) in presence or absence of HG hAdipo-CM. As positive control, the cells were treated with E₂ alone. After 24 hours, cell viability was assessed by sulforhodamine B assay, as described in Methods. For the panels (B), (C) and (D), the results were reported as percentage of viable cells compared with positive control, considered as 100% viable cells. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^*p<0.05; ^***p<0.001); ^# denote statistically significant values compared with tam treatment in absence of (B) hAdipo-CM, (C) adipocytes (^#p<0.05; ^##p<0.01); § denote statistically significant values compared with tam treatment in CTRL_siRNA cells (^§p<0.05).

Figure 6. Effect of adipocyte-released IL8 on MCF7 cell breast tamoxifen responsiveness

(A) Mature adipocytes were incubated in serum free high glucose medium (25mM; HG). After 8 hours, media were collected (HG hAdipo-CM) and applied onto MCF7 cells in presence or absence of IL8 blocking antibody (1μg/ml; IL8-Ab). As control, the cells were incubated in serum-free HG medium. After four days, CTGF mRNA expression levels in MCF7 cells were determined by qRealTime-PCR, as described in Methods. Data were normalized on Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT) gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the expression levels of CTGF compared to those in control cells (dotted line). ^* denote statistically significant value compared with control (^*p<0.05); ^# denote statistically significant values compared with HG hAdipo-CM treatment in absence of IL8-Ab (^#p<0.05). (B) MCF7 cells were treated with estradiol (100nM, E₂) and tamoxifen (5μM; tam) in presence of HG hAdipo-CM, with or without IL8-Ab (1μg/ml; IL8-Ab). As positive control, the cells were treated with E₂ alone. After 4 days, cell viability was determined by sulforhodamine B assay, as described in Methods. The results were reported as percentage of viable cells compared with positive control, considered as 100% viable cells. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^**p<0.01; ^***p<0.001);^# denote statistically significant values compared with tam treatment in presence of HG hAdipo-CM (^#p<0.05). (C) MCF7 cells were incubated in presence or absence of human recombinant IL8 protein (1μg/ml; rIL8). After four days, CTGF mRNA expression levels were determined by qRealTime-PCR, as described in Methods. Data were normalized on HPRT gene as internal standard. Bars represent the mean ± SD of three independent triplicate experiments and show the expression levels of CTGF in cells treated with rIL8 relative to those in untreated cells. ^* denote statistically significant values (^**p<0.01). (D) MCF7 cells were treated with E₂and tam (5μM) in presence or absence of rIL8 (1μg/ml). As positive control, the cells were treated with E₂ alone. After 4 days, cell viability was determined by sulforhodamine B assay (see Methods). The results were reported as percentage of viable cells compared with positive control, considered as 100% viable cells. Data represent the mean ± SD of three independent triplicate experiments. ^* denote statistically significant values compared with positive control (^*p<0.05; ^***p<0.001); ^# denote statistically significant values compared tam treatment in absence of rIL8 (^#p<0.05). See also Supplementary Figures 2 and 3.

Figure 7. CTGF IHC staining in ER⁺ breast cancer tissues and Kaplan-Meier curves

(A) Negative staining of CTGF in cancer cells at 20x and 40x magnification (Upper panels, respectively). Positive staining of CTGF in cancer cells (black arrows) at 20x and 40x magnification (Lower panels, respectively). Details of inflammatory cells CTGF staining (red arrows) represented positive internal control. (B) Kaplan-Meier curves representing overall survival (OS) and disease-free survival (DFS), have been calculated as described in Experimental Procedures. The follow-up duration was five years. Patients which stained positive for CTGF were reported as Green line; patients which stained negative as blue line.

Figure 8. Glucose regulation of BC cell responsiveness to tamoxifen

Glucose reduces tamoxifen responsiveness inducing CTGF expression in BC cells. Glucose promotes the adipocytes’ release of IL8 that in turn impairs BC cell responsiveness to tamoxifen modulating CTGF.