Acquisition of neuronal traits in breast cancer cells demonstrates tumor cell plasticity during metastatic colonization

Mukti R Parikh, Purdue University

Abstract

Metastatic spread to the bone is the most common cause of mortality amongst breast cancer patients. Metastasis is a complex multi-step process whereby tumor cells invade the basement membrane of primary tissue (mammary gland), enter the circulation by traversing the vascular endothelium, and finally extravasate the circulatory system to colonize a site with permissive microenvironment, such as the bone marrow (BM). Tumor cell dissemination and invasion can occur as early as the premalignant phase of mammary tumorigenesis, thus highlighting metastasis as an early event and further emphasizing the necessity to identify the molecular events leading to tumor metastasis. The purpose of this study was to identify the molecular events responsible for tumor metastasis and determine the factors that provide metastatic efficiency to tumor cells. We developed a novel three-dimensional in vitro reconstructed metastasis (rMet) model that incorporates extracellular matrix (ECM) elements characteristic of the primary (breast, prostate or lung) and metastatic (bone marrow, BM) sites. A cytokine-rich liquid interphase separates the primary and distant sites recapitulating circulation. Similar to main events underlying the metastatic cascade, the rMet model fractionated human tumor cell lines into sub-populations with distinct invasive and migratory abilities: 1) a primary tumor-like fraction mainly consisting of non-migratory spheroids; 2) an invasive fraction that invaded through the primary tumor ECM, but failed to acquire anchorage-independence and reach the BM; and 3) a highly migratory BM-colonizing population that invaded the primary ECM, survived in the `circulation-like' media, and successfully invaded and proliferated within BM ECM. BM-colonizing fractions successfully established metastatic lesions in vivo, whereas the tumor-like spheroids were latent in forming metastatic lesions, showing the ability of rMet model to faithfully select for highly aggressive sub-populations with a propensity to colonize a metastatic site. We validated the specificity of the rMet model to study metastasis by using various human breast cancer (MCF7, MDA-MB-231, MDA-MB-231-BO, MCF10CA1a), prostate cancer (LNCaP, PC3) and lung cancer (A549) cell lines in the rMet model. Metastatic MDA-MB-231, MDA-MB-231-BO, PC3, and A549 cells colonized the BM successfully whereas non-metastatic MCF7 and LNCaP cells failed to colonize the BM. Using MCF10CA1a and MCF10AneoT cells, we established a distinct pro-metastatic gene signature for invasive and BM-colonizing cells. The gene signature comprises cell adhesion molecules, ECM remodeling enzymes, stem cell signaling proteins, growth factors, and chemokines. We studied morphologic and phenotypic changes in breast cancer cells during distinct stages of metastasis, a phenomenon also known as tumor cell plasticity. MCF10CA1a breast cancer cells underwent partial epithelial-mesenchymal transitions, as evident from reduced expression of E-cadherin (epithelial) and vimentin (mesenchymal) in the BM-colonizing fraction, and an increased expression of N-cadherin (mesenchymal) in the BM-colonizing fraction. We also found a sub-population within BM-colonizing cells with a morphology, resembling neurons, rather than epithelial or mesenchymal cells, suggesting morphologic plasticity of cells during metastatic colonization. A subset of BM-colonizing cells was also positive for GAP-43 (growth associated protein), NCAM (neural cell adhesion molecule), microtubule associated proteins MAPT (Tau), and MAP1b, all of which are known to play a role in neuronal migration. Using PCR arrays, we showed that genes with a role in neuronal migration, neuron-specific functions such as axonogenesis, and neuron projection development were upregulated in the BM-colonizing fraction. Some examples of upregulated genes included brain derived neurotrophic factor, netrin1, tenascin R, neuronal-glial related cell adhesion molecule, and pleiotrophin, further reflective of the ability of metastatic cells to hijack mechanisms to gain a survival advantage during metastatic colonization. Our study shows that the rMet model recapitulates the complexity of metastatic disease and hence, is a robust system to study metastatic colonization. Identification of novel features of phenotypic and morphologic plasticity during metastasis provides insights into mechanisms and cellular features governing metastatic efficiency to cancer cells and also suggests molecular targets for development of anti-metastatic strategies in the future.

Degree

Ph.D.

Advisors

Kirshner, Purdue University.

Subject Area

Molecular biology|Cellular biology|Systematic|Oncology

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