Effects of peripheral blood mononuclear cells on Haemonchus contortus larval motility in vitro.

The objective of this experiment was to determine effects of peripheral blood mononuclear cells (PBMC), derived from parasite-resistant St. Croix (STC) and parasite-susceptible Suffolk (SF) sheep, on motility of Haemonchus contortus L3 stage larvae in vitro. Peripheral blood mononuclear cells were collected from 10 lambs of each breed, 5 naïve and 5 which had received a priming infection with H. contortus. Larval motility was quantified using a MIF Nikon Sweptfield microscope and NIS Elements AR software, and measurements included path length (PL) (µm), velocity (VEL) (µm/s) and acceleration (ACC) (µm/s2 ). After 18 h of incubation, PL and VEL were greatest in larvae cultured with SF-derived PMBC and were significantly different from all other groups (P < 0·01). No difference was observed in PL or VEL between larvae exposed to naïve or primed STC-derived PBMC and primed-SF PBMC. Differences in ACC were detected between larvae cultured with primed STC-derived PBMC (10·91 µm/s2 ) and naïve SF-derived PBMC (45·7 µm/s2 ) (P = 0·035). These data indicate an innate ability of STC-derived PBMC to severely inhibit larval motility.

Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer.

The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop.

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting that Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src-transformed Abl(-/-)/Arg(-/-) fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib-treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR-invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively, our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.