Sara Duhachek-Muggy

An essential role of metalloprotease-disintegrin ADAM12 in triple-negative breast cancer

In the absence of HER2 overexpression, triple-negative breast cancers (TNBCs) rely on signaling by epidermal growth factor receptor (EGFR/ErbB1/HER1) to convey growth signals and stimulate cell proliferation. Soluble EGF-like ligands are derived from their transmembrane precursors by ADAM proteases, but the identity of the ADAM that is primarily responsible for ligand release and activation of EGFR in TNBCs is not clear. Using publicly available gene expression data for patients with lymph node-negative breast tumors who did not receive systemic treatment, we show that ADAM12L is the only ADAM with an expression level significantly associated with decreased distant metastasis-free survival times. Similar effect was not observed for patients with ER-negative non-TNBCs. There was a positive correlation between ADAM12L and HB-EGF and EGFR in TNBCs, but not in ER-negative non-TNBCs. We further demonstrate that ectopic expression of ADAM12L increased EGFR phosphorylation in a mouse intraductal xenograft model of early breast cancer. Finally, we detect strong correlation between the level of anti-ADAM12L and anti-phospho-EGFR immunostaining in human breast tumors using tissue microarrays. These studies suggest that ADAM12L is the primary protease responsible for the activation of EGFR in early stage, lymph node-negative TNBCs. Thus, our results may provide novel insight into the biology of TNBC.

Metalloproteinase-disintegrin ADAM12 is associated with a breast tumor-initiating cell phenotype

Members of the ADAM family of proteases have been associated with mammary tumorigenesis. Gene profiling of human breast tumors identified several intrinsic subtypes of breast cancer, which differ in terms of their basic biology, response to chemotherapy/radiation, preferential sites of metastasis, and overall patient survival. Whether or not the expression of individual ADAM proteases is linked to a particular subtype of breast cancer and whether the functions of these ADAMs are relevant to the cancer subtype have not been investigated. We analyzed several transcriptomic datasets and found that ADAM12L is specifically up-regulated in claudin-low tumors. These tumors are poorly differentiated, exhibit aggressive characteristics, have molecular signatures of epithelial-to-mesenchymal transition (EMT), and are rich in markers of breast tumor-initiating cells (BTICs). Consistently, we find that ADAM12L, but not the alternative splice variant ADAM12S, is a part of stromal, mammosphere, and EMT gene signatures, which are all associated with BTICs. In patients with estrogen receptor-negative tumors, high expression of ADAM12L, but not ADAM12S, is predictive of resistance to neoadjuvant chemotherapy. Using MCF10DCIS.com breast cancer cells, which express the endogenous ADAM12L and efficiently form mammospheres when plated at the density of single cell per well, we show that ADAM12L plays an important role in supporting mammosphere growth. We postulate that ADAM12L may serve as a novel marker and/or a novel therapeutic target in BTICs.

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

BackgroundADAM12-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.MethodsmiR-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 35S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.ResultsTransfection 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.ConclusionsThe 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.

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

Six 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.

Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer

BackgroundADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells.MethodsADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown.ResultsWe found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown.ConclusionsThese results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.

Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells

Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.

Alternative mRNA Splicing Generates Two Distinct ADAM12 Prodomain Variants

Human ADAM12, transcript variant 1 (later on referred to as Var-1b), present in publicly available databases contains the sequence 5′-GTAATTCTG-3′ at the nucleotide positions 340–348 of the coding region, at the 3′ end of exon 4. The translation product of this variant, ADAM12-Lb, includes the three amino acid motif 114VIL116 in the prodomain. This motif is not conserved in ADAM12 from different species and is not present in other human ADAMs. Currently, it is not clear whether a shorter variant, Var-1a, encoding the protein version without the 114VIL116 motif, ADAM12-La, is expressed in human. In this work, we have established that human mammary epithelial cells and breast cancer cells express both Var-1a and Var-1b transcripts. Importantly, the proteolytic processing and intracellular trafficking of the corresponding ADAM12-La and ADAM12-Lb proteins are different. While ADAM12-La is cleaved and trafficked to the cell surface in a manner similar to ADAM12 in other species, ADAM12-Lb is retained in the ER and is not proteolytically processed. Furthermore, the relative abundance of ADAM12-La and ADAM12-Lb proteins detected in several breast cancer cell lines varies significantly. We conclude that the canonical form of transmembrane ADAM12 is represented by Var-1a/ADAM12-La, rather than Var-1b/ADAM12-Lb currently featured in major sequence databases.

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.

Abstract B43: ADAM12 is a novel regulator of stem-like cells in triple-negative breast cancer

High expression of metalloprotease ADAM12 is associated with increased metastasis in triple-negative breast cancers (TNBC) and is an independent prognostic factor of chemoresistance in TNBC. ADAM12 is up-regulated in the claudin-low subtype of breast tumors and in the mesenchymal stem-like subtype of TNBC. Furthermore, expression of ADAM12 is elevated in post-therapy tumors. The goal of our study was to examine whether ADAM12 may serve as a marker or a therapeutic target in cells with enhanced metastatic cancer stem cell phenotype, using two representative TNBC cell lines with aggressive and metastatic phenotype, SUM159PT and Hs578T. We found that sorted cell subpopulations with the highest cell surface expression of ADAM12 had increased expression of cancer stem cell markers, decreased proliferation rates in 2-D cultures, and increased ability to form mammospheres under suspension conditions. ADAM12 knockdown decreased mammosphere formation, increased apoptotic cell death upon detachment, and decreased cell migration and invasion. ADAM12 knockdown also decreased the population of CD44+/CD24- cells and ALDEFLUOR+ cells and reduced tumor formation in mice in vivo, suggesting that ADAM12 might be necessary for maintaining cancer stem cell phenotype of TNBC cells. To get insight into molecular pathways modulated by ADAM12, we performed RNA sequencing in cells with inducible knockdown of ADAM12 and identified several candidate pathways. Our results indicate that ADAM12 is a novel regulator of the plasticity of TNBC stem cells and it may serve as a future target in anti-metastatic breast cancer therapies. Citation Format: Yue Qi, Sara Duhachek-Muggy, Hui Li, Linda Alyahya, Randi Wise, Anna Zolkiewska. ADAM12 is a novel regulator of stem-like cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B43.

Abstract B133: Metalloprotease-disintegrin ADAM12-L in breast cancer cells: Regulation of expression by microRNA-200b/c

ADAM12-L, a member of the cell surface ADAM metalloprotease family, is selectively up-regulated in claudin-low breast cancers and in residual breast tumors after conventional therapy. In vitro, ADAM12-L is enriched in breast cancer cells grown in suspension as mammospheres and after induction of epithelial-to-mesenchymal transition (EMT) by transforming growth factor β or Twist. The mechanisms responsible for this pattern of ADAM12-L expression are not known. The 39-UTR of ADAM12-L mRNA contains several predicted target sites for miR-200b/c, important regulators of EMT, tumor invasion, metastasis, and chemosensitivity. The mRNA splice variant encoding the secreted isoform ADAM12-S and lacking miRNA-200b/c target sites in the 39UTR shows a different expression pattern than ADAM12-L. The purpose of this study was to determine whether ADAM12-L expression in breast cancer cells is regulated by miR-200b/c. To achieve this goal, breast cancer cells with high expression of ADAM12-L and low levels of miR-200b/c (SUM159PT, SUM1315MO2, and Hs578T) were treated with miR-200b/c mimics. Cells with low expression of ADAM12-L and high levels of miR-200b/c (SUM225PT, SUM149PT, and MCF7) were treated with miR-200b/c inhibitors. To determine whether ADAM12-L mRNA is a direct target of miR-200b/c, we performed co-transfection experiments using miR-200b/c mimics/inhibitors and ADAM12-L 39UTR reporter constructs with intact or mutated miR-200b/c target sites. Our results indicate that miR-200b/c down-regulates ADAM12-L levels in breast cancer cells, but the mechanism does not seem to involve direct targeting of its 39UTR. As ADAM12-L is virtually not present in normal breast tissue, we propose that ADAM12-L may be considered as a novel cell surface marker of tumor cells undergoing EMT and/or as a read-out for miR-200b/c activity in breast tumors. Citation Format: Sara Duhachek-Muggy, Anna Zolkiewska. Metalloprotease-disintegrin ADAM12-L in breast cancer cells: Regulation of expression by microRNA-200b/c. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B133.