MENA Confers Resistance to Paclitaxel in Triple-Negative Breast Cancer

Taxane therapy remains the standard of care for triple-negative breast cancer. However, high frequencies of recurrence and progression in treated patients indicate that metastatic breast cancer cells can acquire resistance to this drug. The actin regulatory protein MENA and particularly its invasive isoform, MENAINV , are established drivers of metastasis. MENAINV expression is significantly correlated with metastasis and poor outcome in human patients with breast cancer. We investigated whether MENA isoforms might play a role in driving resistance to chemotherapeutics. We find that both MENA and MENAINV confer resistance to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MENAINV -driven metastatic lesions. Mechanistically, MENA isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells. MENA expression also increases MAPK signaling in response to paclitaxel treatment. Decreasing ERK phosphorylation by cotreatment with MEK inhibitor restored paclitaxel sensitivity by driving microtubule stabilization in MENA isoform-expressing cells. Our results reveal a novel mechanism of taxane resistance in highly metastatic breast cancer cells and identify a combination therapy to overcome such resistance

Tumor Cell-Driven Extracellular Matrix Remodeling Drives Haptotaxis during Metastatic Progression

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration

UNCONVENTIONAL ROLES OF HUMAN N-MYRISTOYLTRANSFERASES

157 pagesN-myristoyltransferases (NMTs) are ubiquitous among eukaryotes and are widely known for adding a myristoyl, saturated 14-carbon chain, to the amino group of the N-terminal glycine on proteins. This modification in turn regulates essential protein properties such as membrane binding, stability, enzymatic activity, and interactions with other proteins. Decades of elegant studies have established the rules that govern this modification, such as the substrate sequence with the indispensable N-terminal glycine and co-translational mode of action. During my graduate work I discovered functions of NMT that do not conform to the known principles of this enzyme. I began my work with discovering that the small GTPase ADP-ribosylation factor 6 (ARF6) is myristoylated on lysine 3 in addition to its common to the ARF family N-terminal glycine myristoylation. Excitingly, this finding offered an explanation to the puzzling properties of ARF6 compared to ARF1-5 such as its retention at the membrane during the GTPase cycle. We became curious in finding the enzyme that adds this modification, however there was no reported mammalian lysine fatty-acyl transferases. Because N-terminal myristoylation occurs at the amino group, just like lysine myristoylation, my advisor Prof. Hening Lin suggested to test NMT. With the experimental help and suggestions of several amazing scientists, I demonstrated that it was indeed true. I then found that SIRT2 removes this modification, which closed the fascinating myristoylation-demyristoylation cycle essential for ARF6 activation. My following work focused on establishing the role of this cycle in colon cancer progression and on discovering other unconventional substrates of NMT such as Dynamin 2.2022-08-2

MenaINV mediates synergistic cross-talk between signaling pathways driving chemotaxis and haptotaxis

Directed cell migration, a key process in metastasis, arises from the combined influence of multiple processes, including chemotaxis—the directional movement of cells to soluble cues—and haptotaxis—the migration of cells on gradients of substrate-bound factors. However, it is unclear how chemotactic and haptotactic pathways integrate with each other to drive overall cell behavior. MenaINV has been implicated in metastasis by driving chemotaxis via dysregulation of phosphatase PTP1B and more recently in haptotaxis via interaction with integrin α5β1. Here we find that MenaINV-driven haptotaxis on fibronectin (FN) gradients requires intact signaling between α5β1 integrin and the epidermal growth factor receptor (EGFR), which is influenced by PTP1B. Furthermore, we show that MenaINV-driven haptotaxis and ECM reorganization both require the Rab-coupling protein RCP, which mediates α5β1 and EGFR recycling. Finally, MenaINV promotes synergistic migratory response to combined EGF and FN in vitro and in vivo, leading to hyperinvasive phenotypes. Together our data demonstrate that MenaINV is a shared component of multiple prometastatic pathways that amplifies their combined effects, promoting synergistic cross-talk between RTKs and integrins

MenaINV mediates synergistic cross-talk between signaling pathways driving chemotaxis and haptotaxis

Directed cell migration, a key process in metastasis, arises from the combined influence of multiple processes including chemotaxis, the directional movement of cells to soluble cues, and haptotaxis, the migration of cells on gradients of substrate-bound factors. However, it is unclear how chemotactic and haptotactic pathways integrate with each other to drive overall cell behaviour. Mena[superscript INV] has been implicated in metastasis by driving chemotaxis via dysregulation of phosphatase PTP1B, and more recently in haptotaxis via interaction with integrin α5β1. Here we find that Mena[superscript INV]-driven haptotaxis on fibronectin (FN) gradients requires intact signalling between α5β1 integrin and the epidermal growth factor receptor EGFR, which is influenced by PTP1B. Furthermore, we show that Mena[superscript INV]-driven haptotaxis and ECM reorganization both require the Rabcoupling protein RCP, which mediates α5β1 and EGFR recycling. Finally, Mena[superscript INV] promotes synergistic migratory response to combined EGF and FN in vitro and in vivo, leading to hyper-invasive phenotypes. Together, our data demonstrate that Mena[superscript INV] is a shared component of multiple pro-metastatic pathways that amplifies their combined effects, therby promoting synergistic crosstalk between RTKs and integrins.United States. Dept. of Defense. Breast Cancer Research Program (Grant W81XWH-12-1-0031)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Grant U54- CA112967)Kathy and Curt Marble Cancer Research FundNational Cancer Institute (U.S.) (Grant P30-CA14051

Tumor cell-driven extracellular matrix remodeling enables haptotaxis during metastatic progression

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration.Ludwig Center for Molecular OncologyBreast Cancer Research Program (U.S.) (post-doctoral fellowship, W81-XWH-12-1-0031)National Institutes of Health (U.S.) (NIH grant U54-CA112967)National Institutes of Health (U.S.) (NIH grant R01 CA142833)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (NIH grant U01 CA143069)Kathy and Curt Marble Cancer Research Fund (Koch Institute Frontier Award)Prostate Cancer FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (NCI core grant P30-CA14051