Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells

Abstract

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and β-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and β-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Keywords: AXL receptor tyrosine kinase; amoeboid migration; cell aggregation; collective cell migration; metastasis.

Figure 1. Tpm2.1 is downregulated in breast cancer and Tpm2.1-silencing in MCF10A retards wound healing migration independent of 2D substrates

(A) Expression of Tpm isoforms were detected in breast epithelial (MCF10A), breast cancer (MCF7, T47D, BT-474, SK-BR-3, BT-20, MDA-MB-231 and MDA-MB-468) and cervical cancer (HeLa) cell lines against α9d and TM311 antibody. (B) Oncomine (http://www.omcomine.org) data of Tpm2.1 in normal vs metastatic cancer patients. Tested samples are mentioned below the graph; normal versus invasive lobular breast carcinoma (ILC), P-value: 2.26 × 10^-10; fold change: −2.256 and normal versus invasive ductal breast carcinoma (IDC), P-value: 4.57 × 10^-25; fold change: −2.496. (C) MCF10A cells were treated with siRNA against Tpm2.1 for 72 hours and silencing was detected using immunoblotting. α-tubulin was used as a loading control. Error bars indicated means ± s.e.m; *P < 0.05, **P < 0.01 as compared with control, Student's t-test. (D–E) More than three independent experiments of siRNA-treated cells were grown to confluent monolayer on different substrates and quantified by wound closure area by Image J (Scale bar: 100 μm).

Figure 2. Downregulation of Tpm2.1 retards collective cell migration

(A) Protein expression of Tpms were detected after 50, 100 nM RNAi treatment in MCF10A cells. (B) Collective cell migration was monitored at 0 and 15 hours and (C) quantified the area of wound closure using Image J (Scale bar: 100 μm). More than three independent experiments were performed (means ± s.e.m; **P < 0.01, ***P < 0.001; Student's t-test). (D–E) RNAi treated cells were stained against E-cadherin and vinculin together with phalloidin and DAPI under confluent cell state, and at the cell edge of wound, in confluent monolayer. Magnified images are represented for detailed image (Scale bar: 20 μm). (F) Immunoblotting against E-cadherin and vinculin after RNAi treatment in MCF10A cells. α-tubulin was used as a loading control. (G) MCF10A cell clusters were cultured for 24 hours under serum and growth factor starved condition followed by 100 ng/ml EGF treatment to monitor cell scatter (Scale bar: 20 μm). (H) MCF10A cells cultured for 24 hours under serum and growth factor starved condition were wounded and monitored for 12 hours after 100 ng/ml EGF treatment (Scale bar: 20 μm). (I) EGF treated cells, cultured for 24 hours were stained against E-cadherin (yellow box) with phalloidin and DAPI (Scale bar: 20 μm).

Figure 3. Downregulation of Tpm2.1 increases the rate of amoeboid cell migration, invasion and single cell migration

(A–B) MCF10A cells were silenced with Tpm2.1 siRNA and were seeded on PET membranes to measure cell migration or Matrigel-coated membranes to measure invasion. The results represent four independent experiments (means ± s.e.m; ***P < 0.001; Student's t-test). (C) Live cell imaging was used to monitor single cell migration after RNAi treatment on fibronectin coated plates. White and yellow circles indicate cell motility in different time-lapse images (Scale bar: 20 μm).

Figure 4. Tpm2.1 regulates the assembly of adhesion proteins in focal adhesion

(A) Tpm2.1 siRNA-treated MCF10A cells adhered on fibronectin coated cover glass for 4 hours were stained for vinculin, actin filaments using phalloidin and DAPI (Scale bar: 20 μm). (B–C) Cells adhered for 4 hours, immunofluorescence against phospho-paxillin and total-paxillin (Scale bar: 20 μm). Yellow boxes were enlarged for detailed analysis (Inset).

Figure 5. Inhibition of Rho kinase restores motility following downregulation of Tpm2.1

(A) Tpm2.1-silenced MCF10A cells were treated with 10 μM Y27632 or 25 μM blebbistatin and were scratched to make a wound (Scale bar: 100 μm). (B) Quantified data of A. More than three independent experiments were performed. (C) Immunoblotting data against E-cadherin of RNAi treated MCF10A cells followed by DMSO or Y27632 treatment. α-tubulin was used as a loading control. (D–E) The leading edge of cells undergoing wound healing were stained with antibodies against E-cadherin and vinculin, and actin filaments visualized with phalloidin and DAPI after Y27632 treatment of RNAi treated MCF10A cells. (Scale bar: 20 μm).

Figure 6. Downregulation of Tpm2.1 affects expression of AXL, E-cadherin and β-catenin in MCF10A cells

(A) Cell lysates from MCF10A cells cultured under different densities were analyzed by immunoblot for expression of the indicated proteins. (B) Expression of myosin IIA and myosin IIB were analyzed by immunoblot. α-tubulin was used as a loading control.

Figure 7. Downregulation of Tpm2.1 induces more compact spheroids

(A, D) siRNA and shRNA transfected MCF10A cells were cultured in ultra-low attachment 96-well plates to form spheroids. After 96 hours, compact spheroids were formed and Tpms expression were detected by immunoblotting (Scale bar: 500 μm). (B, E) After 96 hours, quantification of the spheroid size were measured by SigmaScan Pro 4.0 and (C, F) Quantification of cell compactness were represented through cell viability by spheroid size. More than three independent experiments were performed (means ± s.e.m; *P < 0.05, ***P < 0.001; Student's t-test).

Figure 8. Rate of migration from spheroid depends on microenvironment

(A) RNAi treated MCF10A cells were re-cultured in non-adherent plate and protein expression of total cell lysates was detected after 96 hours. (B) Spheroids were re-cultured on different substrates coated on culture plates to measure cells migrating out of the spheroids at 48 and 72 hours (Scale bar: 500 μm). (C) Immunofluorescent data for β-catenin, phalloidin and DAPI were imaged at the trailing cell sheet and leading edge of spheroid migration under uncoated culture condition. (D–E) Cells migrating out of the spheroid on collagen I and fibronectin were stained for vinculin, phalloidin and DAPI (Scale bar: 20 μm).