ABSTRACT
Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.
Subject(s)
Antigens, Neoplasm/metabolism , Breast Neoplasms/enzymology , Carbonic Anhydrase IX/metabolism , Cell Movement/physiology , Mammary Neoplasms, Experimental/enzymology , Matrix Metalloproteinase 14/metabolism , Animals , Antigens, Neoplasm/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carbonic Anhydrase IX/genetics , Cell Line, Tumor , Female , HEK293 Cells , Humans , MCF-7 Cells , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Matrix Metalloproteinase 14/genetics , Mice , Podosomes/enzymology , Podosomes/genetics , Podosomes/pathology , TransfectionABSTRACT
B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA, miR-125b-1, is overexpressed in B-ALL cases with the t(11;14)(q24;q32) translocation; therefore, we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting B-cell regulator of immunoglobulin heavy-chain transcription (Bright)/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was downregulated in B-ALL patients with the t(11;14)(q24;q32) translocation. Repression of Bright/ARID3a blocked differentiation and conferred a survival advantage to Ba/F3 cells under interleukin-3 starvation. In addition, overexpression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis by blockade of caspase activation by a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis.
Subject(s)
DNA-Binding Proteins/physiology , MicroRNAs/physiology , Precursor Cells, B-Lymphoid/physiology , Transcription Factors/physiology , Cell Differentiation , Cell Proliferation , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Humans , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cells, B-Lymphoid/cytology , Translocation, Genetic , Tumor Cells, Cultured , Tumor Suppressor Protein p53/physiologySubject(s)
Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 7/genetics , Chromosomes, Human, Pair 9/genetics , Oncogene Proteins, Fusion/genetics , PAX5 Transcription Factor/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/classification , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proteins/genetics , Translocation, Genetic/genetics , Biomarkers, Tumor/blood , Blood Cell Count , Child, Preschool , Cytoskeletal Proteins , Female , Humans , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Transcription FactorsABSTRACT
Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.
