Epidermal-mesenchymal Transition Research Articles

Abstract P3-10-14: LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL

Abstract Background: LIV-1 is an estrogen-inducible gene that has been implicated in epidermal-to-mesenchymal transition (EMT) in preclinical models of progression and metastasis. Its expression is associated with node-positivity in breast cancer; and has been detected in a variety of cancer types, including estrogen receptor positive breast cancers. SGN-LIV1A is a novel antibody drug conjugate targeting LIV-1 that is currently being evaluated in the I-SPY 2 TRIAL. In this pilot study, we evaluated LIV-1 levels by IHC within HR/HER2/MammaPrint (MP) defined subtypes among patients screening for the I-SPY 2 TRIAL and its correlation to microarray assessed LIV-1 expression levels. Method: In a pilot study, LIV-1 IHC staining was performed by Quest Diagnostics on the pre-treatment samples of 38 patients screening for the I-SPY 2 TRIAL. Pre-treatment expression data generated on a custom Agilent 44K platform was also available. We summarized the LIV-1 H-Scores and percent (%)-positivity across the population and within HR/HER2/MP subtypes; and we assessed the Pearson correlation between LIV-1 H-Score and LIV-1 gene expression levels. In addition, we compared the pre-treatment LIV-1 expression levels within HR/HER2/MP subtypes across I-SPY 2 TRIAL patients from completed arms and their relevant controls (n=989) using ANOVA and post-hoc Tukey tests. Our statistics are descriptive rather than inferential; and does not take into account multiplicities of other biomarkers outside of this study. Results: Of the 38 patients evaluated, 37 have LIV-1 %-positivity > 0; and 18 (47%) have 100% LIV1 positivity. The median LIV-1 H-Score is 200; and 89% of patients (34/38) have moderate/high LIV-1 staining (with H-Score≥100). Of the 34 patients who proceeded onto the trial (and have known HR/HER2/MP status), 9 are triple negative, 19 are HR+HER2-, and 6 are HER2+. Due to our small sample size, we did not further subset the triple negative and HER2+ cases; but within the HR+HER2- patients, 10 are MP1 compared to 9 who are MP2 class. LIV1 H-Score appears highest within the HR+HER2-MP1 cases (median: 290), followed by the HER2+ (median: 216), then the HR+HER2-/MP2 (median: 155), and the TN (median: 120) subtype. LIV1 H-score is significantly correlated with LIV-1 mRNA expression levels (Rp=0.79, p<0.0001). Consistent with these observations, LIV-1 pre-treatment expression levels are significantly higher in the HR+HER2-MP1 group relative to all other HR/HER2/MP defined subtypes (Tukey HSD p < 0.0001) across the I-SPY 2 TRIAL population. The HR+HER2+MP1 group also have high LIV-1 expression levels. Conclusion: Our result suggest that although LIV-1 expression differs by subtype, it is expressed at a moderate/high level in the majority of patients. The good correlation between IHC and array-based LIV-1 expression levels enables us to leverage the entire existing I-SPY 2 dataset and confirm the high rates of LIV-1 expression across the I-SPY 2 population. Further studies to evaluate LIV-1 expression as a biomarker of response to LIV-1 targeting therapies for the neoadjuvant treatment of breast cancer are warranted and ongoing in I-SPY 2. Citation Format: Yau C, Brown-Swigart L, Asare S, I-SPY 2 TRIAL Consortium, Esserman L, van' t Veer L, Beckwith H, Forero A, Rugo H. LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-14.

ASR352, A potent anticancer agent: Synthesis, preliminary SAR, and biological activities against colorectal cancer bulk, 5-fluorouracil/oxaliplatin resistant and stem cells

Despite new agent development and short-term benefits in patients with colorectal cancer (CRC), metastatic CRC cure rates have not improved due to high rates of 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX)-resistance and a clinical therapeutic plateau. At the same time, this treatment regime leads to significant toxicity, cost, and patient inconvenience. Drug-resistance is linked to CRC stem cells, which are associated with the epidermal-to-mesenchymal transition (EMT) pathway. Thus, to optimally treat CRC, a therapy that can target the cell survival and EMT pathways in both CRC bulk and stem cell populations is critical. We recently identified a novel small molecule NSC30049 (7a) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC bulk, FOLFOX-resistant, and CRC stem cells both in vitro and in vivo models. In the present study, we report the synthesis and anti-CRC evaluation of several stable and effective 7a analogs. ASR352 (7b) was identified as one of the equipotent 7a analogs that inhibited the growth of CRC bulk cells, sensitized FOLFOX-resistant cells, and reduced the sphere formation capacity of CRC stem cells. It appears that the complex mechanism of cytotoxicity for 7b includes abrogation of 5-FU-induced the S phase, reduction of the phosphorylation of Chk1 at S317P, S345P and S296P, increased γH2AX staining, activation of caspase 3/PARP1 cleavage, and enhancement of Bax/Bcl2 ratio. Further 7b-mediated reduced phosphorylation of Chk1 was an indirect effect, since it did not inhibit Chk1 activity in an in vitro kinase assay. Our findings suggest that 7b as a single agent, or in combination with 5-FU can be developed as a therapeutic agent in CRC bulk, FOLFOX-resistant, and CRC stem cell populations for unmanageable metastatic CRC conditions.

Downregulation of CSN6 attenuates papillary thyroid carcinoma progression by reducing Wnt/β-catenin signaling and sensitizes cancer cells to FH535 therapy.

The incidence of thyroid cancer has increased worldwide at a rate higher than that of any other cancer. CSN6 is overexpressed in many types of cancers, and such expression is linked to oncogenic activity. However, the detailed biological functions of CSN6 in papillary thyroid cancer (PTC) have not been well characterized. We investigated CSN6 expression in PTC specimens and cell lines. We used short‐hairpin RNA‐mediated gene silencing to explore the biological effects of CSN6 depletion in PTC cells. The combined effects of CSN6 silencing and FH535 therapy were assessed in terms of cell viability. The mechanism by which CSN6 regulated β‐catenin expression was also analyzed. CSN6 levels were determined by real‐time polymerase chain reaction (PCR) (mRNA), Western blotting, and immunochemistry (protein). The CCK‐8 and migration assays and orthotopic xenograft transplantation were used to investigate the biological effects of CSN6. We assessed the combined effects of CSN6 silencing and FH535 on cell viability in vitro. We also analyzed the relationship between the CSN6 level and clinical pathological status. CSN6 was overexpressed in human PTCs, and loss of CSN6 attenuated tumor proliferation and migration both in vitro and in vivo. CSN6 stabilized β‐catenin and facilitated the epidermal‐to‐mesenchymal transition (EMT) in PTC cells. CSN6 positively regulated β‐catenin expression in a β‐Trcp‐dependent manner and triggered expression of several EMT‐related genes regulated by β‐catenin. CSN6 silencing sensitized PTC cells to FH535 therapy via downregulation of the Wnt/β–catenin signaling pathway. Finally, in PTC patients, the level of CSN6 was significantly (inversely) correlated with tumor size, the presence of multifocal lesions, and TNM stage. CSN6 overexpression in PTC is a strong indicator of enhanced tumor aggressiveness. CSN6 promotes PTC progression by inducing the EMT. CSN6 knockdown sensitizes PTC cells to FH535 therapy via downregulation of the Wnt/β–catenin signaling pathway.

MiR-182 inhibits the epithelial to mesenchymal transition and metastasis of lung cancer cells by targeting the Met gene.

The microRNA miR-182, belonging to the miR-183 family, is one of the most frequently studied cancer-related oncogenic miRNAs that is dysregulated in various cancer tissues, and it plays a crucial role in tumorigenesis and tumor progression. Studies revealed that miR-182 might function as an oncogenic or tumor suppressor miRNA in different tissues. However, the role of miR-182 in the development of lung cancer remains largely unknown. miR-182 expression in tumor samples from 58 patients, normal lung tissue samples, and lung cancer cell lines were evaluated by qRT-PCR. Survival curves were analyzed using the Kaplan-Meier method and compared with a log-rank test. Our study demonstrated that miR-182 is frequently downregulated in metastatic NSCLC cells compared with primary tumor tissues. Over-expression of miR-182 significantly inhibited the migration and invasion of lung cancer cells and promoted the expression of the epithelial marker (E-cadherin) in addition to reducing the levels of Snail in lung cancer cells. Further studies demonstrated that miR-182 negatively regulated Met via direct binding to the Met 3'-untranslated region (3'-UTR). Furthermore, we found that miR-182 suppressed the phosphorylation of AKT and the nuclear accumulation of Snail, a transcription factor that promotes the epidermal to mesenchymal transition (EMT). Moreover, miR-182 could repress cell migration, invasion, and EMT of lung cancer cells induced by hepatocyte growth factor (HGF). miR-182 might suppress the EMT and metastasis via inactivation of Met/AKT/Snail in non-small cell lung cancer (NSCLC) cells, which implicates miR-182 may be useful as a new therapeutic target in NSCLC.

Entangling Relation of Micro RNA-let7, miRNA-200 and miRNA-125 with Various Cancers.

Involvement of micro RNAs (miRNA) is currently the focus for cancer studies as they effect the post transcriptional expression of different genes. Let-7 family is among the firstly discovered miRNAs that play important role in cell proliferation and dysregulation leading to cell based diseases including cancer. Another family, miRNA-200 prevents transformation of cell to malignant form and tumor formation by interacting with epidermal mesenchymal transition (EMT). Similarly miRNA-125 controls apoptosis and proliferation by affecting multiple genes involved in transcription, immunological defense, resistance against viral and bacterial infections that ultimately leads to cell proliferation, metastasis and finally cancer. All of these micro RNAs are known to be either upregulated or downregulated in various cancers. Current review is focused to elaborate the role of these three families of micro RNAs on different genes that ultimately cause cancer. In conclusion we can say that the miRNAs discussed here are mostly downregulated in various cancers with some exceptions when upregulation of miRNA-125 may be attributed to cancer formation.

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma.

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

Abstract 5424: Therapeutic targeting of KRAS mutation-driven tumor progression in colorectal cancer

Abstract KRAS is the most frequently altered gene in colorectal cancer (CRC), with mutations occurring in 30-40% of colorectal cancer. Although KRAS mutation is associated with poor prognosis and resistance to anti-epidermal growth factor receptor therapy, the mechanism by which it promotes tumor metastasis remains undefined. Our study explored a new hypothesis that targeting the Y-box binding protein-1 (YB-1)-insulin-like growth factor-I receptor (IGF-IR) signaling axis which is downstream of KRAS, with a novel integrin-linked kinase inhibitor, T315, represents a therapeutically relevant strategy to block KRAS mutation-driven tumor progression. In liver metastases from CRC patients, we observed that there was a preponderance (79/108) of over-expression in both YB-1 and IGF-1R by immunohistochemistry. In colon cancer cell lines HCT-116 and SW480, knockdown and over-expression of KRAS affected the protein and mRNA levels of IGF-1R in a dose-dependent manner. But KRAS only affected the protein level and not mRNA of YB-1, thus suggesting that KRAS regulated YB-1 expression at the post-transcriptional level. Manipulation of YB-1 via plasmid overexpression and siRNA affected IGF-1R protein and mRNA levels in a dose-dependent manner, and YB-1 and IGF-1R promoter binding was confirmed via ChIP assay. There was a dose-dependent decrease in YB-1 and IGF-1R protein levels with MEK162, a MEK inhibitor, but not with LY294002m a PI3K inhibitor. This demonstrated that the KRAS regulation of the YB-1- IGFR-1R signaling axis to be via the MEK signaling pathway and not PI3K/Akt. Our novel drug T315 targets YB-1, and was shown to inhibit cell viability and cell migration in a dose-dependent manner. T315 treatment of the colon cancer cells also decreased protein levels of YB-1 and IGF-1R and affected epidermal mesenchymal transition markers such as E-cadherin, vimentin, and snail. These results suggest that a combination of a MEK inhibitor and T315 may be an effective therapeutic strategy to target KRAS mutation driven CRC. Citation Format: Christina Wu, Peng-Chan Lin, Po-Chen Chu, Hsiao-Ching Chuang, Samuel Kulp, Tanios Bekaii-Saab, Ching-Shih Chen. Therapeutic targeting of KRAS mutation-driven tumor progression in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5424. doi:10.1158/1538-7445.AM2015-5424

Rescuing Cell Polarity with Small Molecule Inhibitors of De‐Palmitoylation

Protein palmitoylation is a unique post‐translational modification required for the membrane anchoring and trafficking of numerous regulatory proteins that play key roles in cell growth, polarity, and signaling. In pulse‐chase palmitoylation proteomics studies, we identified the subset of epithelial tumor suppressors as enzymatically regulated palmitoylated proteins in malignant cells. One such protein, Scribble, localizes at the basolateral membrane where it regulates epithelial cell apical‐basolateral polarity, junctional integrity, proliferation, and metastasis. Loss of Scribble at the plasma membrane cooperates with oncogenic Ras or Myc overexpression to drive tumor formation, promote epidermal to mesenchymal transition (EMT), and bypass contact inhibition. Interestingly, overexpression of Snail in polarized cells eliminates Scribble membrane localization. We recently reported a class of in vivo active small selective inhibitors of the acyl protein thioesterases APT1 and APT2. Addition of a small molecule APT2 inhibitor restores Scribble membrane localization and enhances E‐Cadherin expression, overcoming Snail‐mediated repression. Using a combination of x‐ray crystal structures, medicinal chemistry, and mass spectrometry, we present a broadened understanding of thioesterase function and inhibition in cells. These findings highlight APT2 as a primary regulator of cell polarity protein palmitoylation, and highlight a strategy to attenuate oncogenic signaling and restore cell polarity pathways in malignant cells.

Abstract 5301: Rescuing cell polarity with small molecule inhibitors of de-palmitoylation

Abstract Protein palmitoylation is a unique post-translational modification required for the membrane anchoring and trafficking of numerous regulatory proteins that play key roles in cell growth, polarity, and signaling. Using a pulse-chase palmitoylation proteomics approach, we identified the tumor suppressor Scrib as the most dynamic, enzymatically regulated palmitoylated protein in malignant cells. Scrib localizes at the basolateral membrane where it regulates epithelial cell apical-basolateral polarity, junctional integrity, proliferation, and metastasis. Loss of Scrib at the plasma membrane cooperates with oncogenic Ras or Myc overexpression to drive tumor formation, promote epidermal to mesenchymal transition (EMT), and bypass contact inhibition. Interestingly, overexpression of Snail in polarized cells eliminates Scrib membrane localization. We recently reported a class of in vivo active small selective inhibitors of the acyl protein thioesterases APT1 and APT2. Addition of a small molecule APT2 inhibitor restores Scrib membrane localization and enhances E-Cadherin expression, overcoming Snail-mediated repression of E-cad. These findings suggest that thioesterases, predominantly APT2 more than APT1, regulate the palmitoylation of cell polarity proteins, and highlight a strategy to attenuate oncogenic signaling and restore cell polarity pathways in malignant cells. Citation Format: Jeannie L. Hernandez, Dahvid Davda, Jaimeen Majmudar, Kristin J. Labby, Margery Gang, Sirisha Pasupuleti, Makoto Seita, Brent R. Martin. Rescuing cell polarity with small molecule inhibitors of de-palmitoylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5301. doi:10.1158/1538-7445.AM2014-5301

Rescuing cell polarity with selective small molecule inhibitors of de‐palmitoylation (597.2)

Protein palmitoylation is a unique post‐translational modification required for the membrane anchoring and trafficking of numerous regulatory proteins that play key roles in cell growth, polarity, and signaling. In pulse‐chase palmitoylation proteomics studies, we identified the tumor suppressor Scrib as the most dynamic, enzymatically regulated palmitoylated protein in malignant cells. Scrib localizes at the basolateral membrane where it regulates epithelial cell apical‐basolateral polarity, junctional integrity, proliferation, and metastasis. Loss of Scrib at the plasma membrane cooperates with oncogenic Ras or Myc overexpression to drive tumor formation, promote epidermal to mesenchymal transition (EMT), and bypass contact inhibition. Interestingly, overexpression of Snail in polarized cells eliminates Scrib membrane localization. We recently reported a class of in vivo active small selective inhibitors of the acyl protein thioesterases APT1 and APT2. Addition of a small molecule APT2 inhibitor restores Scrib membrane localization and enhances E‐Cadherin expression, overcoming Snail‐mediated repression. These findings suggest that APT2, and not APT1, regulate the palmitoylation of cell polarity proteins, and highlight a strategy to attenuate oncogenic signaling and restore cell polarity pathways in malignant cells.Grant Funding Source: NIH R00CA151460

Low-dose paclitaxel inhibits the induction of epidermal-mesenchymal transition in the human cholangiocarcinoma CCKS-1 cell line

Epidermal-mesenchymal transition (EMT) confers an advantage to cancer cells by improving their invasive capacity and metastatic potential. This phenomenon by which epidermal cells change into mesenchymal cells and therefore acquire a higher ability to automaticity, is considered a key process in cancer development. Transforming growth factor-β (TGF-β) is a significant factor for accelerating EMT through the activation of proteins, including members of the Smad pathway. Furthermore, previous studies have shown that low-dose paclitaxel (PTX) inhibits EMT in certain cell lines, including those of cancer cells. The present study determined whether low-dose PTX was able to inhibit EMT in a human cholangiocarcinoma CCKS-1 cell line that had been treated with TGF-β1. First, the cytotoxic concentration of PTX for the CCKS-1 cells was identified to be ~5 nM by MTT assay and dead cell staining. Therefore, the concentrations of PTX were set as 1 nM, 2.5 nM and 5 nM for the subsequent experiments. In the morphological investigation, the CCKS-1 cells changed into a spindle morphology and became separated by the administration of TGF-β1. However, low-dose PTX inhibited these changes and the morphology resembled the control cells in a dose-dependent manner. Similarly, immunofluorescence and immunoblotting investigations revealed that the CCKS-1 cells expressed mesenchymal markers following the administration of TGF-β1. However, low-dose PTX inhibited the expression of the mesenchymal markers and the CCKS-1 cells expressed the epithelial marker, E-cadherin. In particular, a concentration-dependent effect was observed in the immunoblotting experiments. These results show that PTX may be able to inhibit EMT in cancer cells, depending on the dose concentration.

Practical strategies for suppressing hypoxia-inducible factor activity in cancer therapy

The utility of anti-angiogenic strategies for cancer control is strongly compromised by hypoxia-driven phenotypic changes in cancer cells, which make cancer cells more invasive and more prone to give rise to metastases. A key mediator of this phenotypic shift is the transcription factor hypoxia-inducible factor-1 (HIF-1), which acts directly and indirectly to promote the epidermal-mesenchymal transition, boost cancer invasiveness, increase production of angiogenic factors, and induce chemoresistance. In some cancers, HIF-1 activity is constitutively elevated even in aerobic environments, making the cancer harder to treat and control. Practical strategies for suppressing HIF-1 activation may include the following: inhibiting NF-kappaB activation with salicylic acid and/or silibinin, which should decrease transcription of the HIF-1alpha gene; suppressing translation of HIF-1alpha mRNA with drugs that inhibit mTOR or topoisomerase I; supporting the effective activity of prolyl hydroxylases - which promote proteasomal degradation of HIF-1alpha under aerobic conditions - with antioxidant measures, alpha-ketoglutarate, and possibly dichloroacetate; promoting the O(2)-independent proteasomal degradation of HIF-1alpha with agents that inhibit the chaperone protein Hsp90; and blocking HIF-1 binding to its DNA response elements with anthracyclines. The utility of various combinations of these strategies should be tested in cancer cell cultures and rodent xenograft models; initial efforts in this regard have yielded encouraging results. Comprehensive strategies for suppressing HIF-1 activity can be expected to complement the efficacy of cancer chemotherapy and of effective anti-angiogenic regimens.

GEMCITABINE RESISTANCE IN PANCREATIC CANCER CELLS IS ASSOCIATED WITH INCREASED EXPRESSION OF STEM CELL-LIKE MARKERS AND A CONCOMITANT DOWN-REGULATION OF PTEN AND ACTIVATION OF AKT

Gemcitabine is currently the standard chemotherapeutic for patients with advanced pancreatic cancer. Despite the rapid development of tumor cell resistance, the specific mechanisms of gemcitabine resistance remain largely unknown and may involve highly resilient cancer stem cells. Whether such cells have relevance in chemoresistance in pancreatic tumors remains unknown. Therefore, the objective of our study was to isolate gemcitabine resistant pancreatic tumor cells from more sensitive parental cells and to determine if they possessed stem cell-like properties. Two resistant cell lines, ASPC1 and L3.6pl, were isolated by growing them in increasing concentrations of gemcitabine. Remaining cells demonstrated effective chemoresistance and were subsequently grown in media containing therapeutic concentrations of gemcitabine. When compared to respective parental cells, resistant cells demonstrated morphologic and biochemical properties of Epidermal to Mesenchymal Transition (EMT). In addition, relative to parental cells, these cells expressed an increased level of surface proteins associated with cancer stem cells, including CD44, CD24, and ESA. Recently, decreased expression of PTEN has been associated with self-renewal of cancer stem cells in hematopoietic tumors, but not normal hematopoietic stem cells. We therefore compared expression of PTEN in our gemcitabine sensitive and resistant cells. We observed that PTEN was decreased >2 fold in both ASPC1 resistant cells and L3.6p1 resistant cell lines relative to their respective parental cells. Consistent with a decreased expression of functional PTEN, we found an increase in pAKT expression but not total Akt in the gemcitabine-resistant cells relative to their respective parental cell lines. Collectively, these results suggest that gemcitabine resistance is associated with an increased cancer stem cell-like population and that decreased PTEN might be involved in pancreatic tumor stem cell renewal, in part through de-repression of the Akt survival pathway. Our results further suggest that overcoming gemcitabine resistance may require novel strategies that inhibit pancreatic stem cell renewal.

Amelioration of Radiation-induced Fibrosis: INHIBITION OF TRANSFORMING GROWTH FACTOR-β SIGNALING BY HALOFUGINONE

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.