Altered regulation of BRCA1 exon 11 splicing is associated with breast cancer risk in carriers of BRCA1 pathogenic variants.

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene.

Evaluation of smartphone-based testing to generate exploratory outcome measures in a phase 1 Parkinson's disease clinical trial.

BACKGROUND: Ubiquitous digital technologies such as smartphone sensors promise to fundamentally change biomedical research and treatment monitoring in neurological diseases such as PD, creating a new domain of digital biomarkers. OBJECTIVES: The present study assessed the feasibility, reliability, and validity of smartphone-based digital biomarkers of PD in a clinical trial setting. METHODS: During a 6-month, phase 1b clinical trial with 44 Parkinson participants, and an independent, 45-day study in 35 age-matched healthy controls, participants completed six daily motor active tests (sustained phonation, rest tremor, postural tremor, finger-tapping, balance, and gait), then carried the smartphone during the day (passive monitoring), enabling assessment of, for example, time spent walking and sit-to-stand transitions by gyroscopic and accelerometer data. RESULTS: Adherence was acceptable: Patients completed active testing on average 3.5 of 7 times/week. Sensor-based features showed moderate-to-excellent test-retest reliability (average intraclass correlation coefficient = 0.84). All active and passive features significantly differentiated PD from controls with P < 0.005. All active test features except sustained phonation were significantly related to corresponding International Parkinson and Movement Disorder Society-Sponsored UPRDS clinical severity ratings. On passive monitoring, time spent walking had a significant (P = 0.005) relationship with average postural instability and gait disturbance scores. Of note, for all smartphone active and passive features except postural tremor, the monitoring procedure detected abnormalities even in those Parkinson participants scored as having no signs in the corresponding International Parkinson and Movement Disorder Society-Sponsored UPRDS items at the site visit. CONCLUSIONS: These findings demonstrate the feasibility of smartphone-based digital biomarkers and indicate that smartphone-sensor technologies provide reliable, valid, clinically meaningful, and highly sensitive phenotypic data in Parkinson's disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Irradiation of juvenile, but not adult, mammary gland increases stem cell self-renewal and estrogen receptor negative tumors.

Children exposed to ionizing radiation have a substantially greater breast cancer risk than adults; the mechanism for this strong age dependence is not known. Here we show that pubertal murine mammary glands exposed to sparsely or densely ionizing radiation exhibit enrichment of mammary stem cell and Notch pathways, increased mammary repopulating activity indicative of more stem cells, and propensity to develop estrogen receptor (ER) negative tumors thought to arise from stem cells. We developed a mammary lineage agent-based model (ABM) to evaluate cell inactivation, self-renewal, or dedifferentiation via epithelial-mesenchymal transition (EMT) as mechanisms by which radiation could increase stem cells. ABM rejected cell inactivation and predicted increased self-renewal would only affect juveniles while dedifferentiation could act in both juveniles and adults. To further test self-renewal versus dedifferentiation, we used the MCF10A human mammary epithelial cell line, which recapitulates ductal morphogenesis in humanized fat pads, undergoes EMT in response to radiation and transforming growth factor ß (TGFß) and contains rare stem-like cells that are Let-7c negative or express both basal and luminal cytokeratins. ABM simulation of population dynamics of double cytokeratin cells supported increased self-renewal in irradiated MCF10A treated with TGFß. Radiation-induced Notch concomitant with TGFß was necessary for increased self-renewal of Let-7c negative MCF10A cells but not for EMT, indicating that these are independent processes. Consistent with these data, irradiating adult mice did not increase mammary repopulating activity or ER-negative tumors. These studies suggest that irradiation during puberty transiently increases stem cell self-renewal, which increases susceptibility to developing ER-negative breast cancer.

Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer.

BACKGROUND: Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. RESULTS: The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. CONCLUSIONS: We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Inflammation Mediates the Development of Aggressive Breast Cancer Following Radiotherapy.

PURPOSE: Women treated with radiotherapy before 30 years of age have increased risk of developing breast cancer at an early age. Here, we sought to investigate mechanisms by which radiation promotes aggressive cancer. EXPERIMENTAL DESIGN: The tumor microenvironment (TME) of breast cancers arising in women treated with radiotherapy for Hodgkin lymphoma was compared with that of sporadic breast cancers. To investigate radiation effects on carcinogenesis, we analyzed tumors arising from Trp53-null mammary transplants after irradiation of the target epithelium or host using immunocompetent and incompetent mice, some of which were treated with aspirin. RESULTS: Compared with age-matched specimens of sporadic breast cancer, radiation-preceded breast cancers (RP-BC) were characterized by TME rich in TGFß, cyclooxygenase 2, and myeloid cells, indicative of greater immunosuppression, even when matched for triple-negative status. The mechanism by which radiation impacts TME construction was investigated in carcinomas arising in mice bearing Trp53-null mammary transplants. Immunosuppressive TMEs (iTME) were recapitulated in mice irradiated before transplantation, which implicated systemic immune effects. In nu/nu mice lacking adaptive immunity irradiated before Trp53-null mammary transplantation, cancers also established an iTME, which pointed to a critical role for myeloid cells. Consistent with this, irradiated mammary glands contained more macrophages and human cells cocultured with polarized macrophages underwent dysplastic morphogenesis mediated by IFNγ. Treating mice with low-dose aspirin for 6 months postirradiation prevented establishment of an iTME and resulted in less aggressive tumors. CONCLUSIONS: These data show that radiation acts via nonmutational mechanisms to promote markedly immunosuppressive features of aggressive, RP-BCs.

Multiscale in situ analysis of the role of dyskerin in lung cancer cells.

Dyskerin is one of the three subunits of the telomerase ribonucleoprotein (RNP) complex. Very little is known about the role of dyskerin in the biology of the telomeres in cancer cells. In this study, we use a quantitative, multiscale 3D image-based in situ method and several molecular techniques to show that dyskerin is overexpressed in lung cancer cell lines. Furthermore, we show that dyskerin expression correlates with telomere length both at the cell population level--cells with higher dyskerin expression have short telomeres--and at the single cell level--the shortest telomeres of the cell are spatially associated with areas of concentration of dyskerin proteins. Using this in vitro model, we also show that exogenous increase in dyskerin expression confers resistance to telomere shortening caused by a telomerase inactivating drug. Finally, we show that resistance is achieved by the recovery of telomerase activity associated with dyskerin. In summary, using a novel multiscale image-based in situ method, we show that, in lung cancer cell lines, dyskerin responds to continuous telomere attrition by increasing the telomerase RNP activity, which in turn provides resistance to telomere shortening.

Lack of radiation dose or quality dependence of epithelial-to-mesenchymal transition (EMT) mediated by transforming growth factor ß.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor ß (TGF-ß)-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-ß-mediated EMT. METHODS AND MATERIALS: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-ß (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. RESULTS: E-cadherin was reduced in TGF-ß-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-ß treatment alone. The radiation quality dependence of TGF-ß-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt/atomic mass unit) (56)Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for (56)Fe ion particles' clonogenic survival, TGF-ß-treated HMECs were irradiated with equitoxic 1-Gy (56)Fe ion or 2-Gy (137)Cs radiation in monolayer. Furthermore, TGF-ß-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of (56)Fe ion underwent TGF-ß-mediated EMT even when only one-third of the cells were directly traversed by the particle. CONCLUSIONS: Thus TGF-ß-mediated EMT, like other non-targeted radiation effects, is neither radiation dose nor quality dependent at the doses examined.

Telomeres and telomerase in lung cancer.

Protected telomeres ensure normal chromosomal segregation during mitosis but at the same time can endow genetically abnormal cancer cells with immortality. Telomerase has a pivotal role in telomere protection, both in normal and cancer cells. Understanding the functional interplay between telomere shortening and telomerase expression in cancer cells is of critical importance to elucidating the precise mechanisms by which these cells are able to bypass telomere crisis and become immortal.

Molecular analysis of a multistep lung cancer model induced by chronic inflammation reveals epigenetic regulation of p16 and activation of the DNA damage response pathway.

The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers and genetic alterations. We analyzed markers of DNA damage response (DDR), proliferative stress, and telomeric stress: gamma-H2AX, p16, p53, and TERT. Lung cancer-related epigenetic and genetic alterations, including promoter hypermethylation status of p16(CDKN2A), APC, CDH13, Rassf1, and Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, and c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase and p53 induction. p16 was also induced in early tumorigenic progression and was inactivated in bronchiolar dysplasias and tumors. Remarkably, lack of mutations of Ras and epidermal growth factor receptor, and a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A), CDH13, and APC, but not in Rassf1 and Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer.