Role of extracellular matrix in breast cancer development: a brief update

Epithelial-mesenchymal transition (EMT)

The EMT (process of losing epithelial characteristics and gaining mesenchymal properties) plays a significant role in the progression of tumor and metastasis, involving different transcription factors (TFs) and signals^15–18. This process is characterized by loss of E-cadherin (cell-cell adhesion molecule) and cytokeratins, along with gain of N-cadherin, fibronectin, and vimentin (mesenchymal cell associated proteins) and this is termed cadherin switching (E – cadherin to N – cadherin)¹⁹. The EMT is regulated by different signaling pathways such as TGF-β, notch, and wnt pathway. All these pathways converge to activate the EMT – specific TFs such as Snail (SNAI 1), slug (SNAI 2), Zeb, and Twist which differentially express in cancerous cells to promote EMT^20–22. Snail is a transcriptional repressor of E-cadherin (cell-cell adhesion molecule), and E-cadherin loss is a hallmark of EMT². Snail and TWIST cooperate inducing another TF, ZEB1²³ (significant inducer of EMT, invasion, and metastasis), which is triggered by extracellular hyaluronic acid (HA). Furthermore, ZEB1 induces HAS2 synthesis, promoting HA production in a positive feedback loop and its expression is correlated with ZEB1 expression in poor prognosis tumors. HAS2 also has a role in TGF-β-induced EMT²⁴. Platelets and platelet-derived TGF-β promote epithelial-mesenchymal-like transition and promote metastasis in vivo²⁵. Both the canonical (Smad dependent pathway) and noncanonical (Smad independent pathway) TGF-β signaling activate the TFs (Snail, Zeb, Twist, and Six1) responsible for EMT. The TGF-β induced EMT might be facilitated through enhanced expression of PARP3 (Poly ADP-Ribose Polymerase 3) protein which promotes cell motility, and chemoresistance in breast epithelial cells²⁶. The PARP3 seems to promote stemness of cancerous cells by inducing stem cell markers SOX2 and OCT4, and increasing the population of CD44^high/CD24^low tumor initiating cells. The notch signaling induces the TFs (Snail, Slug, Twist, and Zeb1/Zeb2) by acting through NF-κB, promoting cytokine production and cell survival. The wnt pathway induces Snail, thus down regulates E-cadherin via β-catenin. Besides these signaling, the hypoxic microenvironment also changes the function of mitochondria leading to HIF1 stimulation and subsequently increased expression of Zeb1 required for EMT²². The inflammatory factors such as TNF-α and IL-1β showed to induce plasticity in nontransformed breast epithelial cells (surrounding the transformed tumor cells) by initiating EMT through Snail and Zeb1²⁷. Apart from this, the steroid nuclear receptors such as estrogen receptors, progesterone receptors, glucocorticoid receptors, and mineralocorticoid receptors are also observed to regulate the expression of TFs inducing EMT²⁸. A calcium - dependent phospholipid binding protein such as annexin A2 likely promotes EMT through activation of EGF/EGFR pathway. It is also observed annexin A2 directly binds to STAT3, which is a key EMT inducer up regulating the expression of TFs for EMT²⁹. Aberrant cancer metabolism promotes EMT which further aggravates metabolism (especially glucose metabolism) through Snail and Twist³⁰. One of the TF Runx1 (highly expressed in epithelium) is found to stabilize the mammary epithelial cell (MEC) phenotype thus prevents the EMT³¹. It is observed EMT is activated by ECM stiffness, which induces the release of TWIST1 from its anchor G3BP2 and this TF enters the nucleus and transcriptionally boost the EMT process through integrin clustering and activation³².

The cancer cells need to overcome anoikis (apoptosis due to loss of attachment to ECM) for metastasis event as this is a crucial barrier preventing tumor cell migration to secondary sites. Induction of anoikis occurs through lysosome – mediated down regulation of epidermal growth factor receptors (EGFRs) resulting in the termination of prosurvival signaling. It is observed the depletion of one of the kinases pre-mRNA splicing factor 4 kinase (PRP4K) promotes increased resistance to anoikis through reduced EGFR degradation (after cell detachment from ECM) with increased level of TrkB, vimentin, and ZEB1³³. Anoikis is evaded in ErbB2 expressing cells by multicellular aggregation during ECM detachment. EGFRs are stabilized by this aggregation, which results in ERK/MAPK survival signaling³⁴. EGFRs could be the therapeutic targets to eliminate the ECM-detached cancer cells.

Enzymes in ECM remodeling

Various ECM-remodeling enzymes are induced in BC promoting stem/progenitor signaling pathways and metastasis. The different pathways that are regulated by ECM during remodeling in BC development, are wnt, PI3K/AKT, ERK, JNK, Src-FAK etc^3,11,35. Major ECM proteins induced are fibrillar collagens, fibronectin, specific laminins, proteoglycans, and matricellular proteins and these could be potential drug targets for therapy³⁶. Matrix metalloproteinases (MMPs) degrade ECM proteins promoting invasion and metastasis. The MMP-11 (stromelysin-3) seems facilitating tumor development through apoptosis inhibition. However, it suppresses metastasis in animal models, exhibiting different roles in tumor progression³⁷. β-D mannuronic acid (BDM) is a MMP inhibitor, inhibiting MMP-2 and MMP-9 involved in invasion, metastasis, and angiogenesis³⁸. BDM possesses anti-metastatic activity and inhibits tumor growth by suppressing inflammatory chemokine and tumor–promoting cytokines³⁹. MMP-14 located on the cell surface, is a potential target to stop metastasis and a novel antibody-mediated MMP-14 blockade seems to limit hypoxia and metastasis in triple negative breast cancer (TNBC) models⁴⁰. The progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) exhibited up regulation of MMPs such as MMP-2, 11, 13, and 14 associated with invasion and ECM remodeling⁴¹. The MMPs along with cross-linking enzymes LOX (Lysil oxidase) facilitate collagen maturation, regulate expression and function of soluble factor like TGF-β which ultimately reciprocates through regulation of expression of many ECM proteins and modifying enzymes including LOXs⁴². Lox is a copper-dependent amine oxidase which initiates the intra- and intermolecular collagen crosslinking through oxidative deamination of specific lysine and hydroxylysine residues located in the telopeptide domains⁴³. This crosslinking stiffens the matrix and promotes focal adhesions (Focal adhesion kinase level is increased), integrin clustering, PI3K signaling which ultimately facilitates ErbB2 – dependent breast tumor invasion¹¹. The increased stiffness of matrix (measured by elastography) showed low response to neoadjuvant chemotherapy as compared to patients with soft breast carcinomas^44,45. It is observed women with high mammographic density (MD) are more likely to develop BC as compared to women with low MD⁴⁶. It seems down-regulation of LOXL4 promotes BC growth and lung metastasis in mice⁴⁷. The LOXL2 protein catalyzes cross-linking of ECM components collagen and elastin and is involved in cancer progression and metastasis. The intracellular LOXL2 shows EMT induction and Snail-1 stabilization, and LOXL-2/Snail-1-mediated E-cadherin down-regulation promotes lung metastasis of BC without affecting ECM stiffness⁴⁸. Collagen is the major scaffolding protein in stroma providing tensile strength to the tissue and its metabolism is dysregulated in cancer with increased expression and deposition⁴⁹. The type I collagen is thought to provide barrier against tumor invasion; however enhanced collagen expression is observed with more incidence of metastasis⁵⁰.

The enzyme collagen prolyl hydroxylase, required for collagen synthesis, is over expressed in BC tissues with poor prognosis³. Besides, the enzyme procollagen lysyl hydroxylase-2 involved in collagen synthesis, increases breast tumor stiffness, promotes metastatic tumors in lymph nodes and lungs. Matrix stiffness promotes tumor progression and invasion of ER+ type BC⁵¹. The hardened ECM drives invasion and metastasis through ERK1/2 signal up-regulation and JAK2/STAT5 signal down-regulation. The enzyme heparanase cleaves heparan sulfate, promoting tumor invasion and metastasis. ER stress during chemotherapy enhances the heparanase activity⁵². The MMTV-heparanase mice promoted growth and metastasis of breast tumor cells to lungs suggesting a role for heparanase in BC progression⁵³. Elemene (extract of C urcuma erhizoma plant), is an anticarcinogenic phytochemical showing effects by down-regulating heparanase expression (potential target for heparanase)⁵⁴. The heparin and nanoheparin derivatives show their anti-cancer activities by reducing BC cell proliferation and metastasis⁵⁵. Loss of ECM integrity by plasmin facilitates cancer cell spread and plasmin-induced ECM degradation may be controlled by lipoprotein-A (competitive inhibitor of plasminogen)^56–58. Vitamin C seems to be very important curbing tumor growth, and metastasis as ECM integrity requires vitamin C⁵⁸.

The ECM proteins such as COL I, III, IV, VI, fibronectin, laminin 332, periostin, and vitronectin promote tumor progression and metastasis, whereas the proteins such as DMBT1, and SPARC suppress BC development and metastasis as reviewed by Zhu et al. (2014)³.

Stromal cells in BC development

Different types of stromal cells that inhabit in the tumor microenvironment (TME), are immune cells, fibroblasts, adipocytes, endothelial cells and bonemarrow derived stem cells⁵⁹. Tumor cells recruit tumor-associated macrophages (TAMs), which become proangiogenic by secreting VEGF-A which nourishes tumor cells and build a vessel network for their invasion. Hypoxia also induces macrophages to produce more VEGF and suppress immune response, promoting invasion⁶⁰. Cancer-associated fibroblasts (CAFs) are involved in tumor development, progression, inflammation, metastasis, and build resistance to cancer therapy through secretion of hormones, cytokines, growth factors, etc. and cross-talk with other stromal cells, cancer cells, and ECM. CAFs facilitate the invasion through paracrine signaling with cancer cells. The cross-talk between CAFs and cancer cells enhances IGF1 secretion by CAFs and PAI-1 (Serpine1) activity in cancer cells⁶¹. These two molecules activate RhoA/ROCK signaling in cancer cells which increases cell scattering and invasion. Another study showed CAFs initially assembling an unfolded fibronectin matrix, later remodeled into a dense predominating collagen I matrix driven by MMPs⁶². This remodeling resulted in structural and mechanical changes in the stroma, promoting proangiogenic signaling and breast tumor invasion. CAFs can be potential therapeutic targets in BC⁶³. Cancer cell proliferation and migration is induced by activated fibroblasts derived from endothelial-to-mesenchymal transformation⁶⁴. The cancer associated adipocytes (CAAs) have a significant role in cancer progression, ECM remodeling, phenotype changes of CAFs, and resistance to cancer therapy⁶⁵. They show tumor-modified phenotype with ability to modify cancer cell phenotype favoring metastasis⁶⁶. Comparative gene expression profiling of myoepithelial cells of cancerous (DCIS) and normal breast tissue showed up regulation of several proteases (cathepsin F, K, and L, MMP2, and PRSS19), protease inhibitors (thrombospondin2, SERPING1, cytostatin C and TIMP3), and collagens like COL1A1, COL3A1, COL6A1 in DCIS tissue^67,68.

Various ECM proteins in BC progression

Integrins, the primary receptors of MECs for ECM, act as sensors of epithelial microenvironment. They are the transmembrane glycoproteins present as heterodimers of α- and β- subunits. Total 8 β – subunits dimerize with 18 α – subunits to form around 24 distinct integrins which specifically bind to different ECM proteins⁶⁹. Their altered expression seems to disorganize ECM and promotes metastasis⁷⁰. Increased MEC proliferation occurs due to enhanced activity of integrin signaling (β1-, β5-, and β6- integrins) by co-activating the oncogenes which augment growth factor signaling. The β1 and β3 integrins play crucial role in BC progression and metastasis, hence therapy needs targeting these two integrins at once or their downstream cytokines like FAKs (focal adhesion kinases) and SFKs (Src family kinases) for effective treatment. One of the studies revealed integrin mediated BC invasion through integrin – uPAR (urokinase/plasminogen activator urokinase receptors) signaling which leads to FRA-1 (Fos-related antigen 1) phosphorylation and invasion⁷¹. The ECM protein vitronectin engagement via integrin and uPAR receptors, ends in activation of SRC and MAPK signaling which ultimately enhances FRA-1 phosphorylation. The FRA-1 (a member of AP-1 family of TFs) targets (which promote tumor cell proliferation, invasion and metastasis) include plasminogen activator, MMP-1, MMP-9, Clca2 (Chloride channel accessory2), adenosine receptor A2B, and miR221/222. Protein ECM1 is involved in angiogenesis, promoting TNBC migration and invasion⁷². Protein Hic-5 (focal adhesion scaffold/adaptor protein) promotes mammary duct formation. Focal adhesions of cells are attached to ECM and transduce signals from ECM to cell. Hic-5 is up-regulated in CAFs of BC, involved in EMT and invadopodia (F-actin rich protrusions of cancer cells) formation facilitating invasion, migration and metastasis⁷³. The sustained directionality of tumor cells to a vessel is promoted by a chemotactic gradient of hepatocyte growth factor (HGF) produced from vessel endothelium. This directional streaming is possible by HGF/c-Met signaling pathway between endothelial cells and tumor cells; and c-Met inhibitors could be a potential target to block tumor cell streaming and metastasis⁷⁴.

Tumor microenvironment (TME) as therapeutic target

Clinical trials are going on intensively at present to target the stromal cells of TME for BC therapy in combination with cancerous cell targets, reviewed recently by Bahrami et al. (2018)⁵⁹. To name a few drugs: drugs that target CAFs are chloroquine, metformin (targets lipid metabolism), anti-Met (targets glucose metabolism), celecoxib (COX-2 inhibitor), PD0332991 (cell cycle arrest), XAV 939 (β-catenin pathway inhibitor), SB431542 (TGF-β1receptor kinase inhibitor) etc. The drugs that target the immune cells are denosumab (Treg cell inhibitor), bisphosphonates (TAM inhibitor), indoximod (IDO pathway inhibitor) etc.

An anthracycline such as doxorubicin treatment of BC shows resistance to the drug mediated by ECM proteins as observed in the in vitro model⁷⁵. Hence, probably combinatorial treatment with integrin signaling inhibitors would be more effective in BC therapy. ABL kinase inhibitors like imatinib, nilotinib, and GNF-5 impede the invadopodia formation, decrease ECM degradation, and impair the matrix proteolysis-dependent invasion as observed in the mouse xenograft model⁷⁶.

The cytotoxic T-cells present in the TME kill the tumor cells. However, their infiltration is retarded by various factors. The chemokines such as CXCL-9, -10, -11(production is induced by IFN-γ and chemokine gradient is established) recruit the T-cells in the TME. The tumor cells produce the extracellular galectin-3 (a lectin that binds to glycans of glycoproteins in ECM), which binds to the glycoprotein IFN-γ and prevents it from inducing secretion of above chemokines, thus impedes the cytotoxic T-cell recruitment in the TME⁷⁷. Immunotherapy targeting the galectin-3 would be better strategy to control tumor growth and invasion.

The TAMs (M2 phenotype) are protumoral in nature suppressing the adaptive immunity (suppresses CD8⁺ T cells), promoting angiogenesis, and matrix remodeling. The TAM can be a potential therapeutic target for BC therapy. Besides, the polarity switch from M2 to M1 phenotype (antitumoral function) could be a better strategy for treatment of cancer^78,79.

MMP-14 was found to be a valid target to control tumor progression and metastasis in triple negative breast cancer. MMP-14 blockade by IgG3369 revealed decreased tumor neoangiogenesis and hypoxia⁸⁰. MMP-11 can be a crucial tumor biomarker and a potential target for immunotherapy⁸¹.

High amount of hyaluronic acid (HA) present in TME seems to act as a physical barrier restricting the antibody and immune cell access to tumor cells⁸². It was observed pericellular matrix of HA^high tumor cells restricted NK cell access and antibody- dependent cell mediated cytotoxicity (ADCC). The hyaluronidase (PEGPH20) treatment showed enhanced trastuzumab-dependent ADCC and NK cell mediated tumor growth inhibition in the in vivo system, proving an effective adjunctive therapy for HA^high tumors⁸².

Benias et al. (2018)⁸³ observed presence of fluid-filled interstitial space in the submucosa of many organs (which are subjected to intermittent compression) supported by thick collagen bundles. Similar structures may also present in breast tissues facilitating the metastasis of cancer cells as opposed to dense connective tissues acting as a barrier to migrating cancer cells.