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

Abstract

Annexin A6 (AnxA6), a member of the calcium (Ca 2+ ) 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 improve 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.

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence

“…Similar to other annexins, ANXA6 binds to negatively charged phospholipids, cholesterol, and nucleotides, as well as a large number of proteins in a Ca 2+ -dependent manner to activate cell membranes in a dynamic, reversible, and regulated manner [ 32 , 33 ]. ANXA6 has been widely studied in cancer, but based on the type and degree of malignancy of the tumor, ANXA6 can have tumor-inhibitory activity or tumor-promoting activity [ 34 ]. We performed proteome sequencing by collecting samples from the anterior section of the maximum diameter of the intrarenal aorta in patients with AAA then found that ANXA6 was highly expressed in AAA tissue.…”

Linking Late Endosomal Cholesterol with Cancer Progression and Anticancer Drug Resistance

“…For instance, AnxA6 overexpression promoted PKCα-and p120GAPmediated EGFR and Ras inactivation, respectively, inhibiting anchorage-independent growth of EGFR overexpressing and ER-negative breast cancer cell lines, A431 migration, invasion and A431 xenograft growth. Furthermore, elevated AnxA6 scaffold levels contributed to improving the efficacy of tyrosine kinase inhibitors (TKIs) targeting EGFR to reduce growth, migration, and invasive properties of EGFR overexpressing A431 carcinoma cells [240][241][242]245].…”

“…Tumor promotor breast cancer [246], cervical cancer [251], esophageal cancer [252], melanoma [252], ovarian cancer [253], pancreatic cancer [254][255][256], women's thyroid cancer [257] Tumor suppressor A431 epithelial carcinoma [240][241][242]245], breast cancer (TNBC, EGFR overexpressing and ER-negative) [239][240][241]246], cervical cancer [249], gastric cancer [248], HCC [250] Chemotherapy response TNBC [239,247,258], gastric cancer [259] On the other hand, tumor promoter activities of AnxA6 have also been reported, with AnxA6 displaying pro-invasive functions in invasive breast cancer cells [239,246]. In gastric cancer, AnxA6 conferred drug resistance via β1 integrin and FAK activation [259].…”

“…Importantly, AnxA6 overexpression resulted in LE/Lys-Chol accumulation that resembled loss-of-NPC1 function, interfered with cytoplasmic phospholipase A2-dependent transport of cav-1 to the cell surface, and caused mislocalization and dysfunction of the SNAREs proteins Stx4, SNAP23 and Stx6 [126,127,134,136]. Consequently, like NPC1 deficiency, elevated AnxA6 levels reduced cholesterol-sensitive caveolae formation, FN secretion and integrin recycling [127,134,136], effectively reducing LDL-inducible migration and the invasion of CHO and A431 carcinoma cells [69,134,245].…”

Diverse Roles of Annexin A6 in Triple-Negative Breast Cancer Diagnosis, Prognosis and EGFR-Targeted Therapies

“…In human squamous A431 epithelial carcinoma cells, which overexpress EGFR but lack endogenous AnxA6, stable expression of AnxA6 was associated with reduced cell growth [ 79 ]. The ectopic AnxA6 expression in these cells led to growth arrest in the G1 phase of the cell cycle, longer doubling times, contact inhibition upon confluence and reduced proliferation when cultured in low serum media [ 80 ]. Furthermore, stable AnxA6 expression in A431 xenografts also reduced tumor growth in vivo [ 79 ].…”