β1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression

Abstract

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, β1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

Keywords: ITGB1; breast cancer; cancer stem cell; fascin; integrins; β1 integrin.

Figure 1

a–d: Significant association between fascin and ITGB1 expression in breast cancer samples. (a) Scatter plot showing mRNA expression levels of fascin against ITGB1, which were obtained from the publically available breast cancer patients’ gene expression dataset from The Cancer Genome Atlas (TCGA, n = 530). (b) Scatter plot showing mRNA expression levels of fascin against stemness score, which consist of 100 stem‐associated genes and calculated from the TCGA gene expression dataset (n = 530) as previously described.18 Scatter plots showing the mRNA expression levels of samples that are (c) fascin⁺/ITGB1⁺ (n = 37) or (d) fascin⁻/ITGB1⁻ (n = 41) against stemness score. Pearson correlation coefficients (r) and associated p values (p) for the correlation test is shown. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

a–c: Fascin enhances ITGB1 expression and adhesion of MDA‐MB‐231 breast cancer cells. (a) Western blots showing the level of fascin and ITGB1 expression in fascin⁻ and fascin⁺ cells. ITGB1 and fascin were normalized on GAPDH and the numbers shown below the images indicate the mean fold changes (of 3 independent experiments ± SD) in fascin⁻ in reference to fascin⁺ group. (b) Bar graph obtained from flow cytometry showing mean florescent intensity (MFI) of fascin (top) and ITGB1 (bottom) in fascin⁺ and fascin⁻ cells. (c) Bar graph showing adhesion of the fascin⁺ and fascin⁻ MDA‐MB‐231 cells to different ECM. Absorbance (570 nM) was measured as detailed in the methods and used as an indication of cells’ adhesion to different ECM. The results are mean of 4 independent experiments ± SD.

Figure 3

a and b: ITGB1 expression in MDA‐MB‐231 breast cancer cells is critical for fascin‐mediated adhesion. (a) Western blot showing fascin, ITGB1 and FAK expression following stable knockdown of ITGB1 using specific ShRNA (ShITGB1) or scrambled control (ShCon) in either fascin⁺ or fascin⁻ MDA‐MB‐231 cells. The numbers shown below the images indicate the mean fold changes (of 3 independent experiments ± SD) in reference to fascin⁺ ShCon group. (b) Bar graph showing adhesion of ShITGB1 and ShCon cells in fascin⁺ and fascin⁻ group. Absorbance (570 nM) was measured as in the methods and used as an indication of cells’ adhesion to different ECM. The results are mean of 3 independent experiments ± SD.

Figure 4

a‐c: ITGB1 expression in MDA‐MB‐231 breast cancer cells is vital for fascin‐mediated enhanced self‐renewability. Bar graph showing the number of tumorspheres (500 seeded) and organoids (2000 seeded) formed by fascin⁺ and fascin⁻ MDA‐MB‐231 cells in the presence (ShCon) or absence (ShITGB1) of ITGB1. Primary (top) and secondary (bottom) tumorspheres (a) or organoids (b) are mean of 3 independent experiments ± SD. (c) Number of tumorspheres formed by fascin⁺ and fascin⁻ MDA‐MB‐231 cells in the presence (ShCon) or absence (ShITGB1) of ITGB1 ± 2 μM of FAK inhibitor (FAKi). The results are mean of 2 independent experiments ± SD.

Figure 5

a–d: Fascin enrichment of CSCs in MDA‐MB‐231 breast cancer cells is ITGB1‐dependent. CSC features were assessed in fascin⁺ and fascin⁻ MDA‐MB‐231 cells in the presence (ShCon) or absence (ShITGB1) of ITGB1. Bar graphs showing the (a) percentage of CD44^hi/CD24^lo positive population, (b) number of colony formed, (c) percentage of cell survival post treatment with doxorubicin and (d) MFI of vimentin. The results are mean of at least 3 independent experiments ± SD.

Figure 6

a–c: Significant correlation between fascin and ITGB1 expression in neoadjuvant treated breast cancer patients is associated with shorter survival. (a) Bar graph showing the distribution of fascin and ITGB1 expression in our invasive breast cancer samples (137 cases). Kaplan–Meier survival curves showing overall (b) and disease‐free (c) survival of patients blotted in relation to the distribution of fascin and ITGB1 expression.