Mechanisms of Matrix-Induced Chemoresistance of Breast Cancer Cells-Deciphering Novel Potential Targets for a Cell Sensitization

Abstract

Tumor cell binding to microenvironment components such as collagen type 1 (COL1) attenuates the sensitivity to cytotoxic drugs like cisplatin (CDDP) or mitoxantrone (MX), referred to as cell adhesion mediated drug resistance (CAM-DR). CAM-DR is considered as the onset for resistance mutations, but underlying mechanisms remain elusive. To evaluate CAM-DR as target for sensitization strategies, we analyzed signaling pathways in human estrogen-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cells by western blot, proteome profiler array and TOP-flash assay in presence of COL1. β1-Integrins, known to bind COL1, appear as key for mediating COL1-related resistance in both cell lines that primarily follows FAK/PI3K/AKT pathway in MCF-7, and MAPK pathway in MDA-MB-231 cells. Notably, pCREB is highly elevated in both cell lines. Consequently, blocking these pathways sensitizes the cells evidently to CDDP and MX treatment. Wnt signaling is not relevant in this context. A β1-integrin knockdown of MCF-7 cells (MCF-7-β1-kd) reveals a signaling shift from FAK/PI3K/AKT to MAPK pathway, thus CREB emerges as a promising primary target for sensitization in MDA-MB-231, and secondary target in MCF-7 cells. Concluding, we provide evidence for importance of CAM-DR in breast cancer cells and identify intracellular signaling pathways as targets to sensitize cells for cytotoxicity treatment regimes.

Keywords: CAM-DR; CREB; FAK; MAPK; Wnt signaling; breast cancer; cisplatin; collagen; integrin; mitoxantrone.

Figure 1

β1-Integrin-related signaling pathways and their deregulation by COL1 binding. (A) Schematic illustration of three potential signaling pathways induced by β1-integrin binding to COL1 as potential contribution to increased cell survival. Wnt, FAK/PI3K/AKT, and MAPK pathways are highlighted in purple, red and green, respectively. (B) Data set of a human proteome profiler kinase activity array of MCF-7 cells and (C) of MDA-MB-231 cells cultivated on plain surfaces (‘wt’); cultivated on COL1 (‘COL1’) or treated with the EC₅₀ values of MX when cultivated on COL1 (‘COL1 + MX’). Each sample consists of a cell lysate, standardized to 200 µg protein per membrane. Data refer to a deregulation of FAK (red), CREB (orange), ERK1/2 (green), β-catenin and GSK-3α/β (both purple). Their roles in the respective signaling pathways are indicated in (A). COL1: Collagen type 1, MX: Mitoxantrone.

Figure 2

Investigation of a potential involvement of Wnt signaling in MCF-7 resistance formation. Flow cytometric detection of Wnt components in MCF-7 cells treated with EC₅₀ of MX (A) or CDDP (B) influenced by COL1 or integrin activation by Mn(II). (C) Western blot data of MCF-7 cells treated in the same manner as (A) confirm that β-catenin levels remain unchanged inside the cytosol (cyto.) and the nucleus (nuc.). (D) Detection of Wnt activity in MCF-7 cells by TOP-flash assay. Data indicate that Wnt activity is not affected by COL1 or Mn(II) in response to MX or CDDP. Presenting the mean of at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05, ** p < 0.01.

Figure 3

Western blot data of FAK/PI3K/AKT pathway components in MCF-7 cells and their deregulation by integrin activation and MX cytotoxic treatment. Protein levels of (A) FAK and pFAK; (B) PI3K and pPI3K; (C) AKT and pAKT are displayed normalized to total protein stainfree analysis and in relation to untreated MCF-7 cells as control (CTR, red line for comparison). The samples were treated in-between activation by Mn(II), COL1 or combined Mn(II) and COL1 in absence of MX (grey) or presence of EC₅₀ MX (blue). (D) Shown is a representative Western blot, but all experiments were conducted in at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05, ** p < 0.01.

Figure 4

Inhibition of FAK and PI3K and the impact on MCF-7 sensitivity to MX and CDDP cytotoxicity. (A) Exemplary data of MTT results to clarify calculation of the resistance factor (RF) as ratio of EC₅₀ values of treated (hollow circle) vs. untreated (full circle) control MCF-7 cells (CTR). The RF confirms that (B) Inhibition of FAK in MCF-7 cells by FAK14 at 1 µM increases sensitivity against MX (blue) and CDDP (yellow). (C) Inhibition of PI3K by BEZ235 at 1 nM significantly sensitizes the MCF-7 cells to MX and CDDP precluding the resistance fostering effect by COL1 observed in absence of BEZ235. Data are presented as EC₅₀ ratios of treated cells vs. untreated cells (RF) in each measurement as means of at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

β1-Integrin knockdown in MCF-7 cells. (A) Western blot data confirm the almost complete deletion of β1-integrin in the knockdown cells compared to the scrambled vector control. (B) The scrambled control MCF-7-sc cells display a typical spreading at a COL1 surface, which is completely different for the MCF-7-β1-kd cells (C), Scale bar:50 µm. (D) The MCF-7-β1-kd cells display a higher sensitivity against MX and CDDP cytotoxicity normalized to the scrambled control cells. (E) Upon CDDP treatment, the MCF-7-β1-kd cells show a lower response to COL1 binding with respect to EC₅₀ compared to scrambled control and are less responsive to Mn(II). Data are means of at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Inhibition of MAPK pathway in MDA-MB-231 cells as a sensitizing strategy. (A) Inhibition of MEK and concurrently downstream molecule ERK in MDA-MB-231 cells by U0126 at 5 µM increases sensitivity against MX (blue) and CDDP (yellow). (B) Inhibition of CREB by 666-15 at 100 nM slightly sensitizes the MDA-MB-231 cells to MX and CDDP cytotoxicity, but not to the extent of U0126. Data are presented as EC₅₀ ratios of treated cells vs. untreated cells (RF) in each measurement as means of at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

The role of MAPK pathway in MCF-7 cells to regulate the sensitivity to MX cytotoxicity. (A) Representative western blot data (n = 3) of the MAPK pathway components in MCF-7 cells in relation to integrin activation by COL1 or Mn(II) or combination thereof in absence of MX (left) or in presence of EC₅₀ of MX (right). (B) Deregulation of the indicated MAPK pathway components in MCF-7 scrambled control (sc) or MCF-7-β1-kd cells (β1-kd), illustrated by a representative western blot (n = 3).

Figure 8

MTT based cytotoxicity studies of (A) MX or (B) CDDP in MCF7-sc and MCF-7-β1-kd cells in dependence on COL1 binding and the impact of the MEK inhibitor U0126 at 5 µM, the ERK inhibitor SCH772984 at 250 nM, the CREB inhibitor 666-15 at 100 nM concentrations. Data are presented as EC₅₀ ratios of treated cells vs. untreated cells (RF) normalized to sc cells. Data are means of at least n = 3 (±SEM), asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.