Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation of MYO3A gene in breast cancer cells

Abstract

The potential of a tumor cell to metastasize profoundly depends on its microenvironment, or "niche" interactions with local components. Tumor-associated-macrophages (TAMs) are the most abundant subpopulation of tumor stroma and represent a key component of tumor microenvironment. The dynamic interaction of cancer cells with neighboring TAMs actively drive cancer progression and metastatic transformation through intercellular signaling networks that need better elucidation. Thus, current study was planned for discerning paracrine communication networks operational between TAMs, and breast cancer cells with special reference to cancer cell invasion and dissemination to distant sites. Here, we report role of MIP-1β in enhancing invasive potential of metastatic breast cancer MDA-MB-231 and MDA-MB-468 cells. In addition, the poorly metastatic MCF-7 cells were also rendered invasive by MIP-1β. The MIP-1β-driven cancer cell invasion was dependent on upregulated expression levels of MYO3A gene, which encodes an unconventional myosin super-family protein harboring a kinase domain. Ex ovo study employing Chick-embryo-model and in vivo Syngenic 4T1/BALB/c mice-model further corroborated aforementioned in vitro findings, thereby substantiating their physiological relevance. Concordantly, human breast cancer specimen exhibited significant association between mRNA expression levels of MIP-1β and MYO3A. Both, MIP-1β and MYO3A exhibited positive correlation with MMP9, an established molecular determinant of cancer cell invasion. Higher expression of these genes correlated with poor survival of breast cancer patients. Collectively, these results point toward so far undisclosed MIP-1β/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1β potentiated cancer cell invasion and metastasis via up regulation of MYO3A gene within cancer cells. Our study exposes opportunities for devising potential anti-metastatic strategies for efficient clinical management of breast cancer.

Keywords: Invadopodia; MIP-1β; MMP-9; MYO3A; invasion; migration.

Figure 1.

Invasive breast adenocarcinoma MDA-MB-231 and MDA-MB-468 exhibited intensified focal degradation of pericellular matrix, increased invadopodia formation and poorly metastatic breast cancer MCF-7 cells were rendered invasive in presence of THP-1 macrophages. (A and B) Representative images from the in vitro matrix degradation assay. Cells (MDA-MB 231 and MDA-MB-468) were seeded on Alexa Fluor 633 labeled gelatin (Red) in absence or presence of macrophages (housed in 0.4 μm PET transwell hanging cell culture insert) and maintained for 24 h, followed by fixation, staining with Alexa fluor 488 phalloidin (Green) and mounted in aqueous media containing DAPI (Blue). Compared to mono-cultured MDA-MB-231 and MDA-MB-468 cancer cells [C], the ones that were co-cultured with macrophages [C+M] exhibited enhanced focal degradation of pericellular matrix as indicated by dark area of degraded fluorescent matrix underneath that cell. Bars represent mean invadopodia count/cell from 10 fields per experiment ±SE (*p < 0.05). (C) Compared to monocultured MCF-7 cells [C], the co-cultured MCF-7 cells (macrophages housed in 0.4 μm PET transwell hanging cell culture insert) [C+M] exhibited enhanced focal degradation (dark area of degraded fluorescent matrix underneath that cell) of pericellular matrix. Bars represent mean invadopodia count/cell (by dot count software) from 10 fields per experiment ±SE (*p < 0.05). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer Cells; C+M: Respective cancer cells co-cultured with macrophages.

Figure 2.

Invasive breast adenocarcinoma MDA-MB-231 and MDA-MB-468 and poorly metastatic breast cancer MCF-7 cells exhibited enhanced localization of cortactin to peripheral cellular structures and potentiated invasive activity in presence of THP-1 macrophages. (A, B and C) Macrophages stimulated formation of functional invadopodia in MDA-MB-231, MDA-MB-468 and MCF-7 cells as revealed by enhanced localization of cortactin to peripheral cellular structures and greater number of cortactin positive puncta overlying foci of matrix degradation in cancer cells co-cultured with macrophages [C+M] as compared to cells that were cultured alone [C]. (D, E and F) Representative images from the in vitro cell invasion assay. The matrigel transvasation assay (MDA-MB-231, MDA-MB-468 and MCF-7) revealed a significant increase in the invasive capacity of co-cultured [C+M] (24 h) cancer cells. Bars represent mean invasive cell count ±SE (*p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer Cells; C+M: Respective cancer cells co-cultured with macrophages.

Figure 3.

Macrophage-assisted ECM degradation and invasion by cancer cells is mediated by MIP-1β. (A and B) Representative images showing the effect of anti-human MIP-1β blockade by MIP-1β-neutralizing antibody (MIP-1β NA) or addition of MIP-1β-purified cytokine (MIP-1β) with respect to matrix protrusive activity, and invasion in MDA-MB-231 and MCF-7. IgG serve as isotype antibody control for MIP-1β-neutralizing antibody (MIP-1β NA). Cancer cells (MDA-MB-231 and MCF-7) treated with MIP-1β NA showed decreased activity in matrix protrusive activity and diminished invasion compared to cells that were not treated with MIP-1β NA. Cancer cells treated with MIP-1β-purified cytokine (MIP-1β) showed an increase in matrix protrusive activity and invasion compare to cells that did not treated with MIP-1β. Bars represent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count ±SE (*p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer Cells; C+M: Respective cancer cells co-cultured withMacrophages; C+M(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C+M+MIP-1β NA: Respective cancer cells co-cultured with macrophages treated with MIP-1β-neutralizing antibody; MIP-1β: Respective cancer cells treated with MIP-1β-purified cytokine.

Figure 4.

Macrophage-assisted ECM degradation and invasion by cancer cells is mediated by MIP-1β via its receptor. (A and B) Silencing MIP-1β cognate receptors viz. CCR4 and CCR5 abrogated macrophage induced in vitro ECM degradation, and invasion by cancer cell. Cancer cells (MDA-MB-231 and MCF-7) silenced with CCR4 and CCR5 showed decreased ECM degardation and invasive activity compared to cells having scrambled in both the conditions viz. cancer cells co-cultured with macrophages [C+M] and cultured alone [C]. Bars represent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count ±SE (*p < 0.05.). Silencing was confirmed by protein gel blot. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C+M: Respective cancer cells co-cultured withmacrophages.

Figure 5.

Blockade of MIP-1β function minimized ex ovo dissemination of co-cultured cancer cells from chick chorioallantoic memebrane to chik brain. (A and B) Representatitive results of ex ovo chick chorioallantoic membrane assay for spontaneous metastasis. MIP-1β-neutralizing antibody (MIP-1β NA) mediated in vivo functional blockade of MIP-1β-impeded spontaneous metastasis of co-cultured (with macrophages) breast cancer cells [C + M + MIP-1β NA] compare to cells that were not treated with MIP-1β NA [C + M + IgG and C + M] from chicken chorioallantoic membrane to chicken brain. Bars represent number of fluorescent foci in each group. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C + M: Respective cancer cells co-cultured with macrophages; C + M(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C + M + MIP-1β NA: Respective cancer cells co-cultured with macrophages treated with MIP-1β-neutralizing antibody.

Figure 6.

TAMs-assisted cancer cell invasion via MIP-1β is dependent on upregulation of MYO3A gene with in cancer cells. (A, B and C) Quantitative RT-PCR-based validation of selected genes (through c-DNA-based gene-expression analysis) in mono-cultured [C] and co-cultured (with macrophages) cancer cells [C+M]. Bars represent relative fold change in expression levels ±SE (*p < 0.05). (D, E and F) MYO3A gene exhibited a characteristic MIP-1β-responsive mRNA exprerssion profile. MIP-1β-neutralizing antibody (MIP-1β NA) treated cells showed decrease in MYO3A expression compared to cells that were co-cultured with macrophages [C+M] and/or IgG antibody control [C + M + IgG]. MIP-1β-purified cytokine (MIP-1β) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1β-purified cytokine (C). Bars represent Quantitative RT-PCR relative fold change expression ±SE (*p <0.05.). All the experiments were done in triplicates and repeated at least thrice. (G and H) Blockade of MIP-1β with MIP-1β-neutralizing antibody (MIP-1β NA, Fig. 6G) and its cognate receptor CCR4 and CCR5 silencing (CCR4-siRNA and CCR5-siRNA, Fig. 6H)) downregulated expression levels of MYO3A in breast cancer cells (MDA-MB-231, MDA-MB-468 and MCF-7). MIP-1β-purified cytokine (MIP-1β) enhanced the expression of MYO3A gene in monocultured cancer cells compared to cells that were not treated with MIP-1β-purified cytokine (C) Fig. 6G. All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C + M: Respective cancer cells co-cultured with macrophages; C+M(IgG): Respective cancer cells co-cultured with macrophages treated with isotype antibody control IgG; C + M + MIP-1β NA: Respective cancer cells co-cultured with macrophages treated with MIP-1β-neutralizing antibody; MIP-1β: Respective cancer cells treated with MIP-1β-purified cytokine.

Figure 7.

MYO3A serves as a key cancer cell intrinsic effector for macrophage-assisted cancer cell invasion via MIP-1β. TAMs-assisted cancer cell invasion via MIP-1β is dependent on upregulation of MYO3A gene within cancer cells. (A and B) Macrophage-induced matrix protrusion and invasion by cancer cells was abrogated upon siRNA-mediated silencing of MYO3A gene compare to scrambled in both C (mon-oculture cancer cells) and C + M (cancer cells co-cultured with macrophages). Bars represent mean invadopodia count/cell from 10 fields per experiment and mean invasive cell count ±SE (*p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—C: Respective cancer cells; C+M: Respective cancer cells co-cultured withmacrophages.

Figure 8.

Invasive breast adenocarcinoma MDA-MB-231 silenced with MYO3A exhibited diminished focal degradation of pericellular matrix, diminished invadopodia formation, compared to Scrambled control in presence of MIP-1β-Purified cytokine. Representative images from the in vitro matrix degradation assay. Cells (MDA-MB-231) Scr and silenced with MYO3A siRNA were seeded on Alexa Fluor 633 labeled gelatin (Red) in absence or presence of MIP-1β-purified cytokine (MIP-1β) for 24 h, followed by fixation, staining with Alexa fluor 488 phalloidin (Green) and mounting in aqueous media containing DAPI (Blue). Compared to Scr control MYO3A-directed siRNA, MDA-MB-231 cancer cells did not show any effect of MIP-1β-purified cytokine on focal degradation of pericellular matrix. Bars represent mean invadopodia count/cell from 10 fields per experiment ±SE (*p < 0.05.). All the experiments were done in triplicates and repeated at least thrice. Abbreviations—MIP-1β: Respective cancer cells treated with MIP-1β-purified cytokine.

Figure 9.

Diminished expression of MMP-9 and MYO3A upon neutralizing antibody-mediated blockade of MIP-1β function was followed by reduced cellular burden in lungs and diminished presence of metastatic foci in liver of syngenic 4T1/BALB/c mouse model of breast cancer. (A and B) Anti-mouse MIP-1β goat IgG polyclonal antibody-treated 4T1 tumors expressed much lower invasive potential as revealed by significantly downregulated mRNA expression levels of MYO3A. The mRNA expression levels of MMP-9 gene were significantly downregulated. Bars represent Quantitative RT-PCR relative fold change expression ±SE (*p <0.05). (C): On day 26th post grafting, compared to controls (PBS or isotype control antibody (IgG)), the intratumoral administration of MIP-1β-neutralizing antibody (MIP-1β NA) resulted in reduced cellular burden in lungs and perivascular regions of liver from 4T1/BALB/c mouse models. Lung and liver sections obtained from healthy uninoculated mice served as mock control. Bars represent no. of metastatic lesions ±SE (*p < 0.05.). All the experiments were done in triplicates. All the experiments were done in triplicates. Abbreviations—4T1 tumor (PBS): 4T1-induced tumor treated with PBS; 4T1 tumor (IgG): 4T1-induced tumor treated with isotype control antibody; 4T1 Tumor (MIP-1β NA): 4T1-induced tumor treated with MIP-1β-neutralizing antibody.

Figure 10.

In vivo blockade of MYO3A expression was followed by reduced cellular burden in lungs and diminished presence of metastatic foci in liver of syngenic 4T1/BALB/c mouse model of breast cancer. (A and B) Confirmation of MYO3A silencing by IHC and protein gel blot analysis in mice tumor tissue. (C) On day 26th post grafting, compared to controls (Scr), the intratumoral administration of MYO3A shRNA resulted in regressed tumor volume and reduced cellular burden in lungs and perivascular regions of liver from 4T1/BALB/c mouse models. Lung and liver sections obtained from healthy uninoculated mice served as mock control. Bars represent no. of metastatic lesions ±SE (*p < 0.05.). All the experiments were done in triplicates. Abbreviations—4T1 tumor (SCR): 4T1-induced tumor treated with scrambled shRNA; 4T1 tumor (MYO3A): 4T1-induced tumor treated with MYO3A shRNA.

Figure 11.

Association between fold change (tumor tissue vs. matched normal) in mRNA expression level of MIP-1β, MYO3A, and MMP-9 in human breast cancer specimen. (A and B) Distribution of breast cancer patients (n = `50) with respect to fold change in expression of selected markers and correlation between fold expressions of marker genes of breast cancer patients (n = 50). (C) Association of MYO3A and MIP-1β expression with overall survival. RT-PCR analysis was done in triplicates and repeated at least thrice.

Figure 12.

Schematic representation of the study. Macrophages-derived cytokine MIP-1β enhances the expression of MYO3A in cancer cells that markedly increase ECM degradation and invasive potential of breast cancer cells. These cascade of events culminate into distant metastasis (Fig. 12).