The genetics, structure and function of the M1 aminopeptidase oxytocinase subfamily and their therapeutic potential in immune-mediated disease.

The oxytocinase subfamily of M1 aminopeptidases plays an important role in processing and trimming of peptides for presentation on major histocompatibility (MHC) Class I molecules. Several large-scale genomic studies have identified association of members of this family of enzymes, most notably ERAP1 and ERAP2, with immune-mediated diseases including ankylosing spondylitis, psoriasis and birdshot chorioretinopathy. Much is now known about the genetics of these enzymes and how genetic variants alter their function, but how these variants contribute to disease remains largely unresolved. Here we discuss what is known about their structure and function and highlight some of the knowledge gaps that affect development of drugs targeting these enzymes.

Characterization of a novel breast cancer cell line derived from a metastatic bone lesion of a breast cancer patient.

PURPOSE: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease. METHODS: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor. RESULTS: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44+/CD49f+/EpCAM-, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions. CONCLUSION: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib.

BACKGROUND: Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. MATERIALS AND METHODS: Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. RESULTS: Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. CONCLUSIONS: Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.

Nemo kinase phosphorylates ß-catenin to promote ommatidial rotation and connects core PCP factors to E-cadherin-ß-catenin.

Frizzled planar cell polarity (PCP) signaling regulates cell motility in several tissues, including ommatidial rotation in Drosophila melanogaster. The Nemo kinase (Nlk in vertebrates) has also been linked to cell-motility regulation and ommatidial rotation but its mechanistic role(s) during rotation remain obscure. We show that nemo functions throughout the entire rotation movement, increasing the rotation rate. Genetic and molecular studies indicate that Nemo binds both the core PCP factor complex of Strabismus-Prickle, as well as the E-cadherin-ß-catenin (E-cadherin-Armadillo in Drosophila) complex. These two complexes colocalize and, like Nemo, also promote rotation. Strabismus (also called Vang) binds and stabilizes Nemo asymmetrically within the ommatidial precluster; Nemo and ß-catenin then act synergistically to promote rotation, which is mediated in vivo by Nemo's phosphorylation of ß-catenin. Our data suggest that Nemo serves as a conserved molecular link between core PCP factors and E-cadherin-ß-catenin complexes, promoting cell motility.

Protein tyrosine phosphatase-alpha complexes with the IGF-I receptor and undergoes IGF-I-stimulated tyrosine phosphorylation that mediates cell migration.

Protein tyrosine phosphatase-alpha (PTPalpha) is a widely expressed receptor-type phosphatase that functions in multiple signaling systems. The actions of PTPalpha can be regulated by its phosphorylation on serine and tyrosine residues, although little is known about the conditions that promote PTPalpha phosphorylation. In this study, we tested the ability of several extracellular factors to stimulate PTPalpha tyrosine phosphorylation. The growth factors IGF-I and acidic FGF induced the highest increase in PTPalpha phosphorylation at tyrosine 789, followed by PMA and lysophosphatidic acid, while EGF had little effect. Further investigation of IGF-I-induced PTPalpha tyrosine phosphorylation demonstrated that this occurs through a novel Src family kinase-independent mechanism that does not require focal adhesion kinase, phosphatidylinositol 3-kinase, or MEK. We also show that PTPalpha physically interacts with the IGF-I receptor. In contrast to IGF-I-induced PTPalpha phosphorylation, this association does not require IGF-I. The interaction of PTPalpha and the IGF-I receptor is independent of PTPalpha catalytic activity, and expression of exogenous PTPalpha does not promote IGF-I receptor tyrosine dephosphorylation, indicating that PTPalpha does not act as an IGF-I receptor phosphatase. However, PTPalpha mediates IGF-I signaling, because IGF-I-stimulated fibroblast migration was reduced by approximately 50% in cells lacking PTPalpha or in cells with mutant PTPalpha lacking the tyrosine 789 phosphorylation site. Our results suggest that PTPalpha tyrosine phosphorylation can occur in response to diverse stimuli and can be mediated by various tyrosine kinases. In the case of IGF-I, we propose that IGF-I-induced tyrosine 789 phosphorylation of PTPalpha, possibly catalyzed by the PTPalpha-associated IGF-I receptor tyrosine kinase, is required for efficient cell migration in response to this growth factor.

PRL PTPs: mediators and markers of cancer progression.

Aberrant protein tyrosine phosphorylation resulting from the altered activity of protein tyrosine phosphatases (PTPs) is increasingly being implicated in the genesis and progression of human cancer. Accumulating evidence indicates that the dysregulated expression of members of the phosphatase of regenerating liver (PRL) subgroup of PTPs is linked to these processes. Enhanced expression of the PRLs, notably PRL-1 and PRL-3, promotes the acquisition of cellular properties that confer tumorigenic and metastatic abilities. Up-regulation of PRL-3 is associated with the progression and eventual metastasis of several types of human cancer. Indeed, PRL-3 shows promise as a biomarker and prognostic indicator in colorectal, breast, and gastric cancers. However, the substrates and molecular mechanisms of action of the PRLs have remained elusive. Recent findings indicate that PRLs may function in regulating cell adhesion structures to effect epithelial-mesenchymal transition. The identification of PRL substrates is key to understanding their roles in cancer progression and exploiting their potential as exciting new therapeutic targets for cancer treatment.

PRL-3: a metastasis-associated phosphatase in search of a function.

The molecular and cellular events involved in cancer progression and metastasis remain much less well-defined than those involved in oncogenesis, despite the fact that cell metastasis is the major factor in cancer mortality. Thus, the discovery that the expression of a protein tyrosine phosphatase, protein of regenerating liver-3 (PRL-3), is upregulated in colon cancer metastases provided an exciting indication that the altered regulation of specific protein tyrosine phosphorylation events and signaling pathways might characterize these metastatic cells and/or be key in promoting the tumor-to-metastasis transition in this, and perhaps other, cancers of epithelial origin. However, the cellular substrate(s) of PRL-3 has not been identified, and little is known of PRL-3-mediated cellular signaling pathways. This review illustrates the significance of PRL-3 in promoting metastasis and the importance of determining the endogenous role of PRL-3.