Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Annexin A6 improves anti-migratory and anti-invasive properties of tyrosine kinase inhibitors in EGFR overexpressing human squamous epithelial cells.

Annexin A6 (AnxA6), a member of the calcium (Ca2+ ) and membrane binding annexins, is known to stabilize and establish the formation of multifactorial signaling complexes. At the plasma membrane, AnxA6 is a scaffold for protein kinase Cα (PKCα) and GTPase-activating protein p120GAP to promote downregulation of epidermal growth factor receptor (EGFR) and Ras/mitogen-activated protein kinase (MAPK) signaling. In human squamous A431 epithelial carcinoma cells, which overexpress EGFR, but lack endogenous AnxA6, restoration of AnxA6 expression (A431-A6) promotes PKCα-mediated threonine 654 (T654)-EGFR phosphorylation, which inhibits EGFR tyrosine kinase activity. This is associated with reduced A431-A6 cell growth, but also decreased migration and invasion in wound healing, matrigel, and organotypic matrices. Here, we show that A431-A6 cells display reduced EGFR activity in vivo, with xenograft analysis identifying increased pT654-EGFR levels, but reduced tyrosine EGFR phosphorylation compared to controls. In contrast, PKCα depletion in A431-A6 tumors is associated with strongly reduced pT654 EGFR levels, yet increased EGFR tyrosine phosphorylation and MAPK activity. Moreover, tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib) more effectively inhibit cell viability, clonogenic growth, and wound healing of A431-A6 cells compared to controls. Likewise, the ability of AnxA6 to inhibit A431 motility and invasiveness strongly improves TKI efficacy in matrigel invasion assays. This correlates with a greatly reduced invasion of the surrounding matrix of TKI-treated A431-A6 when cultured in 3D spheroids. Altogether, these findings implicate that elevated AnxA6 scaffold levels contribute to improve TKI-mediated inhibition of growth and migration, but also invasive properties in EGFR overexpressing human squamous epithelial carcinoma.

Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells.

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Annexin A6-A multifunctional scaffold in cell motility.

Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca2+)-dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.

RNA Interference Using c-Myc-Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models.

In this article, we report the development and preclinical validation of combinatorial therapy for treatment of cancers using RNA interference (RNAi). RNAi technology is an attractive approach to silence genes responsible for disease onset and progression. Currently, the critical challenge facing the clinical success of RNAi technology is in the difficulty of delivery of RNAi inducers, due to low transfection efficiency, difficulties of integration into host DNA and unstable expression. Using the macromolecule polyglycidal methacrylate (PGMA) as a platform to graft multiple polyethyleneimine (PEI) chains, we demonstrate effective delivery of small oligos (anti-miRs and mimics) and larger DNAs (encoding shRNAs) in a wide variety of cancer cell lines by successful silencing/activation of their respective target genes. Furthermore, the effectiveness of this therapy was validated for in vivo tumor suppression using two transgenic mouse models; first, tumor growth arrest and increased animal survival was seen in mice bearing Brca2/p53-mutant mammary tumors following daily intratumoral treatment with nanoparticles conjugated to c-Myc shRNA. Second, oral delivery of the conjugate to an Apc-deficient crypt progenitor colon cancer model increased animal survival and returned intestinal tissue to a non-wnt-deregulated state. This study demonstrates, through careful design of nonviral nanoparticles and appropriate selection of therapeutic gene targets, that RNAi technology can be made an affordable and amenable therapy for cancer.