Mathematical analysis of cell survival allows parameter estimation for radiobiological models and selection of an appropriate model. To our knowledge, no rigorous comparisons on the accuracy of various methods used for such analysis have been performed. In this study we compared three methods: 1. maximization of binomial log-likelihood (BLL); 2. minimization of sum of squares (SS); and 3. method 2 using log-transformed data (SSlog). Analysis of Monte Carlo simulated data (A) generated from the linear-quadratic (LQ) model showed that model parameter estimates from the BLL method were more accurate and less affected by “noise” than those from other methods. Analysis of actual breast cancer cell data showed substantial differences among LQ parameters estimated by the three methods (B). To select among radiobiological models, we used: 1. Sample size-corrected Akaike information criterion (AICc), calculated from BLL method-generated log-likelihood values; and 2. Adjusted coefficient of determination (R2), calculated from SS/SSlog method-generated SS values. Analysis of data simulated from the repair-misrepair (RMR) formalism (C) showed that the first approach outperformed the second approach at identifying the true data-generating model. Examples of how the first approach discriminates between several models were explored using actual mouse (H2AX-proficient and -deficient) and human [DNA-dependent protein kinase (DNA-PK)-proficient and -deficient] cell data (D). Based on this work, we concluded that BLL maximization combined with AICc-based model selection constitutes an effective method for analyzing cell survival data.

Background: Radiation-induced skin reactions often lead to itching and pain. While many cytokines bind and activate nociceptors to initiate pain and the precedent itch upon radiation-induced skin desquamation, little is known that radiotherapy-induced itch can arise from the activation of Notch signaling. With regard to the enhanced ransactivation activity of Notch after irradiation and the Notch-induced intense scratching of mice, we anticipated that a downstream effector of Notch mediates the itch sensation. Aim: The purpose of this study was to investigate the role of Notch receptor in mediating radiation-induced itch. Methods: Primary human keratinocytes, HEK293T/17, and A549 cells with or without NICD (Notch intracellular domain) overexpression were applied for analytical assays, including qRT-PCR, SDS-PAGE, immunoprecipitation, and luciferase assays, for delineating the relationship between Notch and Cathepsin Sas well as the effect of Cathepsin S on the itch-inducing stimulus, thymic stromal lymphopoietin (TSLP). Results: Cathepsin S mRNA and protein were upregulated in response to the overexpression of Notch intracellular domains in primary human keratinocytes. A consensus binding site for Notch-regulated transcription factor CBF-1 (C-repeat/dehydration-responsive element binding factor 1) was identified in the promoter region of Cathepsin S. Along with the observation that Cathepsin S immunoprecipitated with PAR2 (protease-activated receptor 2), TSLP Arch Clin Biomed Res 2016; 1 (1): 9-19 10 was found dramatically increased with Cathepsin S in primary human keratinocytes. Conclusions: This study validates that Cathepsin S is a downstream target gene of Notch and mediates itch by upregulating TSLP. The found mechanism may be targeted for improving patients’ quality of life during radiotherapy.

Purpose: Eribulin mesylate is a novel non-taxane microtubule dynamics inhibitor that irreversibly induces mitotic arrest, and has been shown to promote a shift from mesenchymal to epithelial phenotypes in breast cancer. It has demonstrated therapeutic activity in heavily pretreated patients with metastatic breast cancer, with a trend toward greater efficacy in patients with triple negative breast cancer (TNBC). TNBC is an aggressive tumor with a mesenchymal phenotype and higher locoregional recurrence (LRR) compared to estrogen receptor (ER) positive breast cancer. One postulated cause for higher LRR in TNBC is that these tumors have a great proportion of cancer stem cells (CSC). CSCs are radio- and chemo-resistant when compared to the bulk of the tumor, and are thought to be the source of both LRR and distant metastasis after treatment, thus an important target for therapy. Microtubule inhibitors are known radiation sensitizers given that they arrest cells at G2/M, the most radiation sensitive phase of the cell cycle. We hypothesize that because eribulin induces an irreversible mitotic arrest and promotes a mesenchymal-to-epithelial transition (MET) it will enhance the radiation sensitivity of TNBC through reduction in CSCs and inhibition of DNA damage repair (DDR) pathways. Methods: Experiments were conducted in 3 subtypes of TNBC cells, all with p53 mutations and PTEN loss: SUM149 (basal B), BT-549 (mesenchymal-like), and MDA-MB-468 (basal A). Cells were treated with 0.5nM-3nM Eribulin, alone or in combination with increasing doses of radiation from 0-8 Gy. In vitro studies preformed included; cell survival assays, immunoblot analysis for markers of MET, FACS analysis for cell cycle, cancer stem cell population, and apoptosis, and finally P-γH2AX staining to measure DDR. Results: We found that combining ionizing radiation (IR) and eribulin is synergistic. The IC50 of eribulin combined with 6Gy IR, was 0.5-1 nM, 10 fold less than drug alone, and a 40-60% decrease in cell viability was observed with the combination compared to IR alone. A significant decrease in clonogenic survival was seen after 24 hours pretreatment with 1nM eribulin, with the greatest efficacy seen in BT-549 cells. No significant increase in apoptosis was seen with the combination treatment by FACS analysis though a significant delay in DDR was observed. Cell cycle analysis demonstrated eribulin increases the proportion of cells in G2/M arrest following IR, as well as suppressed cell proliferation. 24-hour eribulin treatment also caused morphological changes in culture consistent with transition from a mesenchymal to an epithelial phenotype, and IHC showed a decrease in mesenchymal markers and increase in epithelial markers. Finally eribulin treatment decreased the proportion of CSCs by FACS analysis by 80% in SUM149 cells and prevented an increase in CSC population following IR. Studies are on going to further characterize the effect of combined eribulin and IR on DDR and the CSC population in TNBC cells. Conclusions: These studies demonstrate that eribulin effectively radiosensitizes TNBC cells. We believe this occurs through inhibition of DDR as well as promotion of MET which reduces the radiation resistant CSC population. Citation Format: Eileen P Connolly, Youping Sun, Tom K Hei. Eribulin mesylate promotes a mesenchymal-epithelial transition (MET) and effectively radio-sensitizes triple negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-04.

Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER)-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα) in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2), correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC) irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM) activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the post-translational maturation of Notch receptors.

Both canonical Wnt/β-catenin and TGFβ/Smad signaling pathways coordinately regulate pattern formation during embryogenesis as well as tumor progression. Evidence of cross-talk between these two pathways has been reported. Here we demonstrated that the Activin-like kinase 4 (Alk4)/Smad2 pathway facilitates the transcriptional activity of the oncogenic Wnt/β-catenin/Tcf4 pathway through a novel Smad4-independent mechanism. Upon activation, Smad2 physically interacted with Tcf4, β-catenin and the co-activator p300 to enhance transcriptional activity of β-catenin/Tcf4 through the histone acetyltransferase activity of p300. Transactivation by Smad2 was independent of a Smad-binding element (SBE) and Smad4. Indeed, the enhancement of β-catenin/Tcf4 transcriptional activity by activated Smad2 was negatively regulated by the presence of Smad4. Moreover, a tumor-derived missense mutant of Smad2, lacking the ability to bind to Smad4 was still able to enhance the Tcf4 transcriptional reporter in the presence of β-catenin and Tcf4. Our findings suggest that Smad2 may function as an activator of canonical Wnt/β-catenin/Tcf4 signaling through a SBE/Smad4-independent pathway.

BACKGROUND:Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis.METHODS:We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided.RESULTS:CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004).CONCLUSION:CR-1 appears to have an important role in the multistep process of angiogenesis.

Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals.

Members of the transforming growth factor-β (TGF-β) family of ligands exhibit potent growth-suppressive and/or apoptosis-inducing effects on different types of cells. They perform essential roles in the elimination of damaged or abnormal cells from healthy tissues. On the other hand, TGF-βs have also been shown to act as tumor-promoting cytokines in a number of malignancies that are capable of stimulating extracellular matrix production, cell migration, invasion, angiogenesis, and immune suppression. Dissecting the complex, multifaceted roles of different TGF-β-related peptides especially during the development of pathological conditions and in carcinogenesis is an area of continuous research and development. The characterization of EGF-CFC proteins as essential co-receptors that contribute to the modulation of the physiological activities of some of the TGF-β ligands will be beneficial for future medical research and the adaptation and possible readjustment of currently applied therapeutic regimes.

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

The neuronal guidance molecule, Netrin-1, has been suggested to play a role in the adhesion and migration of the mammary gland epithelium. Human and mouse Cripto-1 induce proliferation, migration, invasion and colony formation by epithelial cells in 3D matrices. Here we investigate whether Netrin-1 affects these Cripto-1-dependent activities in mouse mammary epithelial cells. Overexpression of Cripto-1 in EpH4 and HC-11 cells (EpH4/Cripto-1 or HC-11/Cripto-1) was associated with low expression of Netrin-1 and increased expression of its receptor Neogenin compared to that of wild-type cells. No change was observed in the expression of the other Netrin-1 receptor, UNC5H1. Treating EpH4/Cripto-1 or HC-11/Cripto-1 mammary cells with exogenous soluble Netrin-1 resulted in increased expression of E-cadherin and UNC5H1, decreased expression of vimentin and decreased activation of Akt as determined by western blotting. Colony formation by Eph4/Cripto-1 cells in 3D gels was significantly reduced in proximity to a Netrin-1 source, and mammary glands of transgenic mice overexpressing human Cripto-1 showed altered ductal growth in proximity to implanted Netrin-1-releasing pellets. Terminal end buds in the treated transgenic mice mammary glands also showed increased expression of E-cadherin and UNC5H1 and decreased expression of active Akt determined by immunohistochemistry. Together, these results suggest that regulation of Netrin-1 expression is important in regulating Cripto-1-dependent invasion and migration of mammary epithelial cells.

Epithelial‐mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto‐1 (CR‐1), a member of the epidermal growth factor‐CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR‐1 transgene by the MMTV promoter (MMTV‐CR‐1) and in mouse mammary epithelial cell line HC‐11 overexpressing CR‐1 (HC‐11/CR‐1). Western blot analysis showed decreased expression of E‐cadherin in MMTV‐CR‐1 tumors and in HC‐11/CR‐1 cells. The expression of N‐cadherin, vimentin, cyclin‐D1, and of the zinc‐finger transcription factor, snail, was increased in MMTV‐CR‐1 tumors. Increased snail mRNA was also found in HC‐11/CR‐1 cells. Expression of phosphorylated (P)‐c‐Src, P‐focal adhesion kinase (FAK), P‐Akt, P‐glycogen synthease kinase 3β (GSK‐3β), dephosphorylated (DP)‐β‐catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV‐CR‐1 tumors. Immunohistochemistry showed positive staining for vimentin, N‐cadherin, cyclin‐D1, smooth muscle actin, fibronectin, snail, and β‐catenin in MMTV‐CR‐1 tumor sections. HC‐11/CR‐1 cells treated with the c‐Src inhibitor PP2 reduced the expression of P‐c‐Src and of P‐FAK, P‐Akt, P‐GSK‐3β, DP‐β‐catenin all known to be activated by c‐Src. Migration of HC‐11/CR‐1 cells was also reduced by PP2 treatment. These results suggest that CR‐1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT. © 2004 Wiley‐Liss, Inc.

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.

Human Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor beta-related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membrane-associated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells.

Human Cripto-1 (CR-1) is a member of the epidermal growth factorCripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor -related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membraneassociated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells.

Using β- and γ-casein mRNAs, the relationship between poly(A) tail length and half-life of mRNA is determined in the mouse mammary gland during pregnancy and lactation. β- and γ-Casein mRNAs increase before and after parturition, respectively. The poly(A) tail as well as the half-life of casein mRNA becomes longer upon the active casein mRNA synthesis. The poly(A) tail is shortened gradually as lactation progresses. The half-life of mRNA decreases approximately from 20 h at early to 4 h at late lactation. Northern blot analysis reveals that nuclear RNA has the same poly(A) tail length as casein mRNA in the cytoplasm does. Thus, the mammary gland changes the poly(A) tail length of casein mRNA. The poly(A) tail length changes in parallel with the level of poly(A) polymerase (PAP) mRNA during pregnancy and lactation, suggesting that the mammary gland determines the poly(A) tail length of casein mRNA through the change in the PAP gene expression. As the half-life of casein mRNA is related with the degree of polyadenylation, we conclude that the poly(A) tail elongation and shortening is a mechanism in regulating the mRNA decay.