ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer

Citation: Duhachek-Muggy, S., & Zolkiewska, A. (2015). ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer. Bmc Cancer, 15, 13. doi:10.1186/s12885-015-1108-1ADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3'-untranslated regions (3' UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3' UTR and they may negatively regulate ADAM12-L expression. Methods: miR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3' UTR by miRNAs was tested using an ADAM12-L 3' UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with S-35 cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors. Results: Transfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3' UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression. Conclusions: The ADAM12-L 3' UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer

The Cysteine-Rich Domain of Human Adam 12 Supports Cell Adhesion through Syndecans and Triggers Signaling Events That Lead to β1 Integrin–Dependent Cell Spreading

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin β1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking β1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain–mediated cell adhesion, and then the β1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn2+ or the β1 integrin–activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12–syndecan complex fails to modulate the function of β1 integrin

Phenotypic diversity of breast cancer-related mutations in metalloproteinase-disintegrin ADAM12

Citation: Qi Y, Duhachek-Muggy S, Li H, Zolkiewska A (2014) Phenotypic Diversity of Breast Cancer-Related Mutations in Metalloproteinase-Disintegrin ADAM12. PLOS ONE 9(3): e92536. https://doi.org/10.1371/journal.pone.0092536Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease

Characterization of human torsinA and its dystonia-associated mutant form.

Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAdeltaE). We found that the signal sequence of torsinA (residues 1-20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24-Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAdeltaE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAdeltaE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells

ADAM12 abrogation alters immune cell infiltration and improves response to checkpoint blockade therapy in the T11 murine model of triple-negative breast cancer

ABSTRACTImmunosuppressive tumor microenvironment (TME) impedes anti-tumor immune responses and contributes to immunotherapy resistance in triple-negative breast cancer (TNBC). ADAM12, a member of cell surface metalloproteases, is selectively upregulated in mesenchymal/claudin-low TNBCs, where its expression is largely restricted to tumor cells. The role of cancer cell-expressed ADAM12 in modulating the immune TME is not known. We show that Adam12 knockout in the T11 mouse syngeneic transplantation model of claudin-low TNBC leads to decreased numbers of tumor-infiltrating neutrophils (TINs)/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and increased numbers of tumor-infiltrating B cells and T cells. ADAM12 loss in cancer cells increases chemotaxis of B cells in vitro and this effect is eliminated by inhibition of CXCR4, a receptor for CXCL12, or anti-CXCL12 blocking antibody. Importantly, ADAM12 loss in T11 cancer cells sensitizes tumors to anti-PD1/anti-CTLA4 combination therapy, although the initial responsiveness is followed by acquired therapy resistance. Depletion of B cells in mice eliminates the improved response to immune checkpoint blockade of Adam12 knockout T11 tumors. Analysis of gene expression data for claudin-low TNBCs from the METABRIC patient cohort shows significant inverse correlations between ADAM12 and gene expression signatures of several anti-tumor immune cell populations, as well as a significant positive correlation between ADAM12 and gene expression signature of TINs/PMN-MDSCs. Collectively, these results implicate ADAM12 in immunosuppression within the TME in TNBC

Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In Vitro

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated “reserve cells” that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin α7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation

The ¹¹⁴VIL¹¹⁶ motif is not conserved between different human ADAMs and between ADAM12 from different species.

<p>Multiple sequence alignment of the region in ADAM proteins flanking the ¹¹⁴VIL¹¹⁶ motif. GenBank accession numbers are: ADAM2, NP_001455; ADAM7, NP_003808; ADAM8, NP_001100; ADAM9, NP_003807; ADAM10, NP_001101; ADAM11, NP_002381; ADAM12, NP_003465; ADAM15, AAS72997; ADAM17, NP_003174; ADAM18, NP_055052; ADAM19, NP_150377; ADAM20, NP_003805; ADAM21, NP_003804; ADAM22, NP_068369; ADAM23, NP_003803; ADAM28, NP_055080; ADAM29, NP_055084; ADAM30, NP_068566; ADAM32, NP_659441; ADAM33, NP_079496; Mouse, NP_031426; Rat, XP_001054670; Cow, NP_001001156; Horse, XP_001490097; Chicken, NP_001136322; Xenopus, NP_00035103. The dog sequence was obtained from e!Ensembl (ENSCAFP00000041414) due to the lack of a signal peptide in the GenBank sequence. Conservation strength is shown in red (high), orange (medium), yellow (poor), and white (no conservation).</p

Breast cancer-associated mutations in human ADAM12-L.

<p>(A) A diagram of human ADAM12-L. Six non-synonymous mutations identified in human breast cancers are indicated. S, signal peptide; P, prodomain; M, metalloproteinase domain; D, disintegrin domain; C, cysteine-rich domain; E, EGF-like domain; T, transmembrane region; Cyt, cytoplasmic tail. (B) Model of the extracellular domain of human ADAM12-L generated by the I-TASSER protein structure prediction tool (C-score <i>−</i>0.26, estimated TM accuracy of the model 0.68±0.12 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092536#pone.0092536-Roy2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092536#pone.0092536-Zhang1" target="_blank">[21]</a>). The metalloproteinase, disintegrin, cysteine-rich, and EGF-like domains are shown in purple, blue, yellow, and green, respectively. Positions of the five amino acids mutated in breast cancers (red spheres) and the side chain of the catalytic residue E351 (cyan sticks) are indicated. (C) Sequence alignment of the cysteine-rich and EGF-like domains of ADAM12 from different species. NCBI RefSeq numbers are: <i>Homo_sapiens</i>, NP_003465; <i>Mus_musculus</i>, NP_031426; <i>Rattus_norvegicus</i>, XP_001054670; <i>Bos_taurus</i>, NP_001001156, <i>Equus_caballus</i>, XP_001490097; <i>Gallus_gallus</i>, NP_001136322, and <i>Xenopus_tropicalis</i>, NP_001035103. (D) Sequence alignment of the cysteine-rich and EGF-like domains of human ADAM12 and the most closely related human ADAMs. NCBI RefSeq numbers are: ADAM19, XP_005266060, ADAM33, NP_079496.1, and ADAM15, NP_997080. In C and D, asterisks indicate three novel mutations in human ADAM12 found in breast tumors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092536#pone.0092536-Shah1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092536#pone.0092536-Jiao1" target="_blank">[15]</a>. Clustal X color scheme was applied.</p