Multiple isoforms of ADAM12 in breast cancer: differential regulation of expression and unique roles in cancer progression

Doctor of PhilosophyDepartment of Biochemistry and Molecular BiophysicsAnna ZolkiewskaThe ADAM (A Disintegrin and Metalloprotease) family of multi-domain proteins modulates a number of cellular signaling pathways in both normal and cancerous cells. ADAM12 has been shown to be a candidate cancer gene for breast cancer and its expression is up-regulated in breast tumors. The human ADAM12 transcript is alternatively spliced. One of these splice variants encodes a transmembrane ADAM12 isoform, ADAM12-L, which has been demonstrated to release cell signaling molecules from the cell surface. Another variant encodes a secreted protease, ADAM12-S, which cleaves extracellular matrix proteins and other secreted proteins. Although these variants are expressed from the same promoter, their relative expression levels are highly discordant. Here, I demonstrate variant-specific regulation of ADAM12 transcripts by microRNAs. Members of the microRNA-29 and microRNA-200 families target the unique 3’UTR of the ADAM12-L transcript and cause transcript degradation. Additionally, I show the presence of a novel ADAM12 splicing event in which 9 additional nucleotides are inserted in the region encoding the autoinhibitory pro-domain. I demonstrate that this novel variant is expressed in breast epithelial cells and breast cancer cell lines. The resulting protein isoform does not undergo proteolytic processing to activate the protease. Additionally, trafficking of the novel isoform to the cell surface is impaired and this isoform is localized to the endoplasmic reticulum. Finally, I determined a role for ADAM12-L in the progression of triple negative breast cancers (TNBCs). These tumors are lacking expression of hormone receptors and the HER2 receptor. HER2 is a member of the epidermal growth factor receptor (EGFR) family and the loss of the HER2 receptor causes tumors to rely on EGFR for propagating pro-growth signals. I show here that, in TNBC tumors, ADAM12-L expression is strongly correlated with poor patient prognosis and increased activation of EGFR. These data suggest that in TNBCs, ADAM12-L enhances tumor growth via EGFR activation. Collectively, the data presented here demonstrate (a) transcript-specific regulation of ADAM12 in breast cancer, (b) the existence of a novel splice variant and protein isoform with impaired cellular trafficking, and (c) an important role of the ADAM12-L isoform in EGFR activation in TNBC

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

Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen

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

Growth Differentiation Factor 11 does not Mitigate the Lethal Effects of Total-Abdominal Irradiation

Total-body exposure to radiation causes widespread tissue injury. Damage to the hematopoietic and intestinal stem cell compartments is particularly lethal and mitigators of this damage are critical in providing effective treatment. Parabiosis radiation experiments, in which the vasculatures of two rodents are anastomosed prior to irradiation of one of the animals, have shown that there is a circulating factor that protects mice from radiation-induced intestinal death. Recently reported studies have suggested that growth differentiation factor 11 (GDF11) is responsible for the rejuvenation of stem cells observed in parabiosis experiments involving aging mice. In this study, we investigated the efficacy of GDF11 as a potential mitigator of radiation-induced damage to intestinal stem cells. In ex vivo cultures of intestinal organoids, the number of cells expressing the stem cell marker Lgr5 was increased after irradiation and GDF11 supplementation. Further ex vivo studies to assess stem cell function, measured by the ability to grow new crypt-like structures, did not show increased stem cell activity in response to GDF11 treatment. In addition, GDF11 was unable to improve survival of mice subjected to total-abdominal irradiation. These data demonstrate that GDF11 does not mitigate radiation damage to intestinal stem cells

ADAM12-Lb is localized to the endoplasmic reticulum.

<p>MCF10A cells stably expressing ADAM12-La or ADAM12-Lb were fixed, permeabilized, and stained with anti- ADAM12 antibody (A, D, G, J, M, P), anti-KDEL antibody (an endoplasmic reticulum marker; B, E), anti-TGN38 antibody (a trans-Golgi marker; H, K), anti-EEA1 antibody (an early endosomal marker; N, Q), and DAPI. Overlay images are shown in panels C, F, I, L, O, R. Partial co-localization of ADAM12-La and EEA1 is indicated by white arrows. Bar, 20 µm.</p

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