β1 Integrins Show Specific Association with CD98 Protein in Low Density Membranes

Background: The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear. Results: We demonstrate that CD98 constitutively and specifically associates with β1 integrins (α2β1,α3β1, α5β1 and α6β1), but minimally with α4β1. Integrin-CD98 association was established by reciprocal immunoprecipitation experiments, and confirmed by CD98-induced clustering of α3β1 but not α4β1 on the surface of rhabdomyosarcoma cells. Integrin-CD98 association is independent of the α subunit cytoplasmic tail, is maintained in α3β1 ligand-interaction deficient mutants, and is not inhibited by EDTA. Within the CD98 heavy chain, a C109S mutation (but not a C330S mutation) caused a loss of β1 integrin association. The same C109S mutation also caused a loss of CD98 light chain association. Importantly, CD98 associated selectively with β1 integrins present in low density "light membrane" fractions on a sucrose gradient. CD98 was not present in dense fractions that contained the majority of β1 integrins. Notably, the C109S mutant of CD98, that did not associate with β1 integrins, showed also a reduced localization into light membrane fractions. Conclusions: We demonstrate that CD98 association with β1 integrins is specific, occurs in the context of low density membranes, and may require the CD98 light chain

Presentation of Integrins on Leukocyte Microvilli: A Role for the Extracellular Domain in Determining Membrane Localization

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins α4β7, αLβ2, and αMβ2 were heterologously expressed in K562 cells. α4β7 initiated adhesion under flow and localized to microvilli, whereas β2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the α4β7 cytoplasmic and/or transmembrane domains with the homologous domains of αLβ2 or αMβ2. Unexpectedly, these chimeras efficiently mediated adhesion to the α4β7 ligand mucosal addressin cell adhesion molecule–1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of α4 and β2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins α4β1, α5β1, α6Aβ1, αvβ3, and αEβ7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli

Development and characterisation of a 3D multi-cellular in vitro model of normal human breast: a tool for cancer initiation studies.

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation

Profiling of Tumour-Infiltrating Lymphocytes and Tumour-Associated Macrophages in Ovarian Epithelial Cancer:Relation to Tumour Characteristics and Impact on Prognosis

Early evidence suggests a strong impact of tumour-infiltrating lymphocytes (TILs) on both the prognosis and clinical behaviour of ovarian cancer. Proven associations, however, have not yet translated to successful immunotherapies and further work in the field is urgently needed. We aimed to analyse the tumour microenvironment of a well-characterised cohort of ovarian cancer samples. Tumour markers were selected owing to their comparative underrepresentation in the current literature. Paraffin-embedded, formalin-fixed tumour tissue blocks of 138 patients representative of the population and including early stage disease were identified, stained for CD3, CD20, CD68 and CD163 and analysed for both the stromal and intertumoral components. Data were statistically analysed in relation to clinical details, histological subtype, borderline vs. malignant status, survival and management received. Mean stromal CD3, total CD3 count, mean stromal CD20 and total CD20 count all correlated negatively with survival. Malignant ovarian tumours consistently demonstrated significantly higher infiltration of all analysed immune cells than borderline tumours. Assessment of the stromal compartment produced a considerably higher proportion of significant results when compared to the intra-tumoural infiltrates. Customary assessment of solely intra-tumoural cells in advanced stage disease patients undergoing primary debulking surgery should be challenged, with recommendations for future scoring systems provided

The calpain system is associated with survival of breast cancer patients with large but operable inflammatory and non-inflammatory tumours treated with neoadjuvant chemotherapy

The calpains are a family of intracellular cysteine proteases that function in a variety of important cellular functions, including cell signalling, motility, apoptosis and survival. In early invasive breast cancer expression of calpain-1, calpain-2 and their inhibitor, calpastatin, have been associated with clinical outcome and clinicopathological factors. The expression of calpain-1, calpain-2 and calpastatin was determined using immunohistochemistry on core biopsy samples, in a cohort of large but operable inflammatory and non-inflammatory primary breast cancer patients treated with neoadjuvant chemotherapy. Information on treatment and prognostic variables together with long-term clinical follow-up was available for these patients. Diagnostic pre-chemotherapy core biopsy samples and surgically excised specimens were available for analysis. Expression of calpastatin, calpain-1 or calpain-2 in the core biopsies was not associated with breast cancer specific survival in the total patient cohort; however, in patients with non-inflammatory breast cancer, high calpastatin expression was significantly associated with adverse breast cancer-specific survival (P=0.035), as was low calpain-2 expression (P=0.031). Low calpastatin expression was significantly associated with adverse breast cancer-specific survival of the inflammatory breast cancer patients (P=0.020), as was low calpain-1 expression (P=0.003). In conclusion, high calpain-2 and low calpastatin expression is associated with improved breast cancer-specific survival in non-inflammatory large but operable primary breast cancer treated with neoadjuvant chemotherapy. In inflammatory cases, high calpain-1 and high calpastatin expression is associated with improved breast cancer-specific survival. Determining the expression of these proteins may be of clinical relevance. Further validation, in multi-centre cohorts of breast cancer patients treated with neoadjuvant chemotherapy, is warranted