The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma.

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

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.

PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

PURPOSE: Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radiation (IR) on PK-M2-mediated metabolic changes in TNBC cells, and their survival. In addition, we determine the effect of PK-M2 activators on breast cancer stem cells, a radioresistant subpopulation of breast cancer stem cells. METHODS: Glucose uptake, lactate production, and glutamine consumption were assessed. The cellular localization of PK-M2 was evaluated by western blot and confocal microscopy. The small molecule activator of PK-M2, TEPP46, was used to promote its pyruvate kinase function. Finally, effects on cancer stem cell were evaluated via sphere forming capacity. RESULTS: Exposure of TNBC cells to IR increased their glucose uptake and lactate production. As expected, PK-M2 expression levels also increased, especially in the nucleus, although overall pyruvate kinase activity was decreased. PK-M2 nuclear localization was shown to be associated with breast cancer stem cells, and activation of PK-M2 by TEPP46 depleted this population. CONCLUSIONS: Radiotherapy can induce metabolic changes in TNBC cells, and these changes seem to be mediated, at least in part by PK-M2. Importantly, our results show that activators of PK-M2 can deplete breast cancer stem cells in vitro. This study supports the idea of combining PK-M2 activators with radiation to enhance the effect of radiotherapy in resistant cancers, such as TNBC.

1-(4-nitrobenzenesulfonyl)-4-penylpiperazine increases the number of Peyer's patch-associated regenerating crypts in the small intestines after radiation injury.

OBJECTIVE: Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS: C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS: Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly, 96 h after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION: In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.

Serum erythropoietin levels, breast cancer and breast cancer-initiating cells.

BACKGROUND: Cancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation. METHODS: Using four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment. RESULTS: Our results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells. CONCLUSIONS: We conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer.

PURPOSE: The lack of a molecular target in triple-negative breast cancer (TNBC) makes it one of the most challenging breast cancers to treat. Radiation therapy (RT) is an important treatment modality for managing breast cancer; however, we previously showed that RT can also reprogram a fraction of the surviving breast cancer cells into breast cancer-initiating cells (BCICs), which are thought to contribute to disease recurrence. In this study, we characterize mebendazole (MBZ) as a drug with potential to prevent the occurrence of radiation-induced reprogramming and improve the effect of RT in patients with TNBC. METHODS AND MATERIALS: A high-throughput screen was used to identify drugs that prevented radiation-induced conversion of TNBC cells into cells with a cancer-initiating phenotype and exhibited significant toxicity toward TNBC cells. MBZ was one of the drug hits that fulfilled these criteria. In additional studies, we used BCIC markers and mammosphere-forming assays to investigate the effect of MBZ on the BCIC population. Staining with propidium iodide, annexin-V, and γ-H2AX was used to determine the effect of MBZ on cell cycle, apoptosis, and double-strand breaks. Finally, the potential for MBZ to enhance the effect of RT in TNBC was evaluated in vitro and in vivo. RESULTS: MBZ efficiently depletes the BCIC pool and prevents the ionizing radiation-induced conversion of breast cancer cells into therapy-resistant BCICs. In addition, MBZ arrests cells in the G2/M phase of the cell cycle and causes double-strand breaks and apoptosis. MBZ sensitizes TNBC cells to ionizing radiation in vitro and in vivo, resulting in improved tumor control in a human xenograft model of TNBC. CONCLUSIONS: The data presented in this study support the repurposing of MBZ as a combination treatment with RT in patients with TNBC.

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.

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

BACKGROUND: ADAM12 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. METHODS: ADAM12 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. RESULTS: We 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. CONCLUSIONS: These 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.

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

BACKGROUND: ADAM12-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 (35)S 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.

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.

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 (114)VIL(116) 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 (114)VIL(116) 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.

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.

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.

Metalloprotease-disintegrin ADAM12 expression is regulated by Notch signaling via microRNA-29.

Metalloprotease-disintegrin ADAM12 is overexpressed and frequently mutated in breast cancer. We report here that ADAM12 expression in cultured mammalian cells is up-regulated by Notch signals. Expression of a constitutively active form of Notch1 in murine fibroblasts, myoblasts, or mammary epithelial cells or activation of the endogenous Notch signaling by co-culture with ligand-expressing cells increases ADAM12 protein and mRNA levels. Up-regulation of ADAM12 expression by Notch requires new transcription, is activated in a CSL-dependent manner, and is abolished upon inhibition of IκB kinase. Expression of a constitutively active Notch1 in NIH3T3 cells increases the stability of Adam12 mRNA. We further show that the microRNA-29 family, which has a predicted conserved site in the 3'-untranslated region of mouse Adam12, plays a critical role in mediating the stimulatory effect of Notch on ADAM12 expression. In human cells, Notch up-regulates the expression of the long form, but not the short form, of ADAM12 containing a divergent 3'-untranslated mRNA region. These studies uncover a novel paradigm in Notch signaling and establish Adam12 as a Notch-related gene.

The role of SnoN in transforming growth factor beta1-induced expression of metalloprotease-disintegrin ADAM12.

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.