Irineu Illa-Bochaca

Radiation Acts on the Microenvironment to Affect Breast Carcinogenesis by Distinct Mechanisms that Decrease Cancer Latency and Affect Tumor Type

Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from tumors arising in nonirradiated hosts. Molecular and genetic approaches show that TGFβ mediated tumor acceleration. Tumor molecular signatures implicated TGFβ, and genetically reducing TGFβ abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGFβ independent and linked to mammary stem cell activity. Thus, the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms.

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 β (TGFβ) 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 TGFβ. Radiation‐induced Notch concomitant with TGFβ 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. Stem Cells 2014;32:649–661

Mapping mammary gland architecture using multi-scale in situ analysis

We have built a novel computational microscopy platform that integrates image acquisition, storage, processing and analysis to study cell populations in situ. This platform allows high-content studies where multiple features are measured and linked at multiple scales. We used this approach to study the cellular composition and architecture of the mouse mammary gland by quantitatively tracking the distribution and type, position, proliferative state, and hormone receptor status of epithelial cells that incorporated bromodeoxyuridine while undergoing DNA synthesis during puberty and retained this label in the adult gland as a function of tissue structure. Immunofluorescence was used to identify label-retaining cells, as well as epithelial cells expressing the proteins progesterone receptor and P63. Only 3.6% of luminal cells were label-retaining cells, the majority of which did not express the progesterone receptor. Multi-scale in situ analysis revealed that luminal label-retaining cells have a distinct nuclear morphology, are enriched 3.4-fold in large ducts, and are distributed asymmetrically across the tissue. We postulated that LRC enriched in the ventral mammary gland represent progenitor cells. Epithelial cells isolated from the ventral versus the dorsal portion of the gland were enriched for the putative stem cell markers CD24 and CD49f as measured by fluorescence activated cell sorting. Thus, quantitative analysis of the cellular composition of the mammary epithelium across spatial scales identified a previously unrecognized architecture in which the ventral-most, large ducts contain a reservoir of undifferentiated, putative stem cells.

Limiting-Dilution Transplantation Assays in Mammary Stem Cell Studies

Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8-10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps.

Insulin-like growth factor-I inhibition with pasireotide decreases cell proliferation and increases apoptosis in pre-malignant lesions of the breast: a phase 1 proof of principle trial

IntroductionEstrogen inhibition is effective in preventing breast cancer in only up to 50% of women with precancerous lesions and many experience side effects that are poorly tolerated. As insulin-like growth factor I (IGF-I) underlies both estrogen and progesterone actions and has other direct effects on mammary development and carcinogenesis, we hypothesized that IGF-I inhibition might provide a novel approach for breast cancer chemoprevention.MethodsIn total, 13 women with core breast biopsies diagnostic of atypical hyperplasia (AH) were treated for 10 days with pasireotide, a somatostatin analog which uniquely inhibits IGF-I action in the mammary gland. They then had excision biopsies. 12 patients also had proliferative lesions and one a ductal carcinoma in situ (DCIS). Primary outcomes were changes in cell proliferation and apoptosis after treatment. Expression of estrogen receptor (ER), progesterone receptor (PR), and phosphorylated Insulin-like growth factor I receptor (IGF-1R), protein kinase B (AKT) and extracellular signal-regulated kinases 1/2 (ERK1/2) were also assessed. Core and excision biopsies from 14 untreated patients served as non-blinded controls. Hyperglycemia and other side effects were carefully monitored.ResultsPasireotide decreased proliferation and increased apoptosis in all AH (from 3.6 ± 2.6% to 1.3 ± 1.2% and from 0.3 ± 0.2% to 1.5 ± 1.6%, respectively) and proliferative lesions (from 3.8 ± 2.5% to 1.8 ± 1.8% and from 0.3 ± 0.2% to 1.3 ± 0.6%, respectively). The DCIS responded similarly. ER and PR were not affected by pasireotide, while IGF-1R, ERK1/2 and AKT phosphorylation decreased significantly. In contrast, tissue from untreated controls showed no change in cell proliferation or phosphorylation of IGF-1R, AKT or ERK 1/2. Mild to moderate hyperglycemia associated with reduced insulin levels was found. Glucose fell into the normal range after discontinuing treatment. Pasireotide was well tolerated and did not cause symptoms of estrogen deprivation.ConclusionsIGF-I inhibition by pasireotide, acting through the IGF-1R, was associated with decreased proliferation and increased apoptosis in pre-malignant breast lesions and one DCIS. Assuming hyperglycemia can be controlled, these data suggest that inhibiting the IGF-I pathway may prove an effective alternative for breast cancer chemoprevention.Trial registrationNCT01372644 Trial date: July 1, 2007.

Notch signaling regulates metabolic heterogeneity in glioblastoma stem cells

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.

In Situ Analysis of Cell Populations: Long-Term Label-Retaining Cells

The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to identify these cells within their tissue context. Here, we introduce long-term label retention as a method to investigate the location of quiescent cells (a property attributed to adult stem cells) in situ. Long-term label retaining cells divide actively during tissue development and remain quiescent at homeostasis. These two properties have been attributed to adult stem cells. Therefore, label-retaining cells can be used to identify populations that contain stem cells. We describe the materials and methods necessary to identify and image mammary label-retaining cells, to carry out morphometric analysis on these cells and to map their distribution of the mammary epithelium. The morphometric and spatial analyses described here are generally applicable to any mammary cell populations, and will therefore be useful to characterize mammary stem cells once bona fide mammary stem cell markers become available.

Systems biology perspectives on the carcinogenic potential of radiation

This review focuses on recent experimental and modeling studies that attempt to define the physiological context in which high linear energy transfer (LET) radiation increases epithelial cancer risk and the efficiency with which it does so. Radiation carcinogenesis is a two-compartment problem: ionizing radiation can alter genomic sequence as a result of damage due to targeted effects (TE) from the interaction of energy and DNA; it can also alter phenotype and multicellular interactions that contribute to cancer by poorly understood non-targeted effects (NTE). Rather than being secondary to DNA damage and mutations that can initiate cancer, radiation NTE create the critical context in which to promote cancer. Systems biology modeling using comprehensive experimental data that integrates different levels of biological organization and time-scales is a means of identifying the key processes underlying the carcinogenic potential of high-LET radiation. We hypothesize that inflammation is a key process, and thus cancer susceptibility will depend on specific genetic predisposition to the type and duration of this response. Systems genetics using novel mouse models can be used to identify such determinants of susceptibility to cancer in radiation sensitive tissues following high-LET radiation. Improved understanding of radiation carcinogenesis achieved by defining the relative contribution of NTE carcinogenic effects and identifying the genetic determinants of the high-LET cancer susceptibility will help reduce uncertainties in radiation risk assessment.

Densely Ionizing Radiation Acts via the Microenvironment to Promote Aggressive Trp53-Null Mammary Carcinomas

Densely ionizing radiation, which is present in the space radiation environment and used in radiation oncology, has potentially greater carcinogenic effect compared with sparsely ionizing radiation that is prevalent on earth. Here, we used a radiation chimera in which mice were exposed to densely ionizing 350 MeV/amu Si-particles, γ-radiation, or sham-irradiated and transplanted 3 days later with syngeneic Trp53-null mammary fragments. Trp53-null tumors arising in mice irradiated with Si-particles had a shorter median time to appearance and grew faster once detected compared with those in sham-irradiated or γ-irradiated mice. Tumors were further classified by markers keratin 8/18 (K18, KRT18), keratin 14 (K14, KRT14) and estrogen receptor (ER, ESR1), and expression profiling. Most tumors arising in sham-irradiated hosts were comprised of both K18- and K14-positive cells (K14/18) while those tumors arising in irradiated hosts were mostly K18. Keratin staining was significantly associated with ER status: K14/18 tumors were predominantly ER-positive, whereas K18 tumors were predominantly ER-negative. Genes differentially expressed in K18 tumors compared with K14/18 tumor were associated with ERBB2 and KRAS, metastasis, and loss of E-cadherin. Consistent with this, K18 tumors tended to grow faster and be more metastatic than K14/18 tumors, however, K18 tumors in particle-irradiated mice grew significantly larger and were more metastatic compared with sham-irradiated mice. An expression profile that distinguished K18 tumors arising in particle-irradiated mice compared with sham-irradiated mice was enriched in mammary stem cell, stroma, and Notch signaling genes. These data suggest that carcinogenic effects of densely ionizing radiation are mediated by the microenvironment, which elicits more aggressive tumors compared with similar tumors arising in sham-irradiated hosts.

A TGFβ–miR-182–BRCA1 axis controls the mammary differentiation hierarchy

TGFβ promotes luminal differentiation of mammary stem cells by suppressing a BRCA1-targeting microRNA. TGFβ directs mammary lineage Breast cancers are often be classified by lineage markers for specific cell types, such as the luminal subtype or the relatively more aggressive myoepithelial (also called basal-like) subtype. Mutations in the protein BRCA1 are associated with the basal-like subtype. The growth factor TGFβ suppresses tumorigenesis in various tissues, but malignant mammary epithelial cells have decreased sensitivity to TGFβ. Using mouse models, Martinez-Ruiz et al. found that loss of TGFβ promoted mammary stem cell self-renewal and skewed differentiation to myoepithelial cells through increased abundance of a microRNA (miR-182) that targets and decreases the translation of BRCA1 transcripts. Expressing BRCA1 or blocking miR-182 restored lineage commitment homeostasis in TGFβ-deficient mammary epithelial cells. The findings link two factors in breast cancer development and may have wider implications for tumorigenesis in other epithelial, ductal tissues (such as the colon, pancreas, and prostate) in which BRCA mutations are also a risk factor. Maintenance of mammary functional capacity during cycles of proliferation and regression depends on appropriate cell fate decisions of mammary progenitor cells to populate an epithelium consisting of secretory luminal cells and contractile myoepithelial cells. It is well established that transforming growth factor–β (TGFβ) restricts mammary epithelial cell proliferation and that sensitivity to TGFβ is decreased in breast cancer. We show that TGFβ also exerts control of mammary progenitor self-renewal and lineage commitment decisions by stringent regulation of breast cancer associated 1 (BRCA1), which controls stem cell self-renewal and lineage commitment. Either genetic depletion of Tgfb1 or transient blockade of TGFβ increased self-renewal of mammary progenitor cells in mice, cultured primary mammary epithelial cells, and also skewed lineage commitment toward the myoepithelial fate. TGFβ stabilized the abundance of BRCA1 by reducing the abundance of microRNA-182 (miR-182). Ectopic expression of BRCA1 or antagonism of miR-182 in cultured TGFβ-deficient mammary epithelial cells restored luminal lineage commitment. These findings reveal that TGFβ modulation of BRCA1 directs mammary epithelial cell fate and, because stem or progenitor cells are thought to be the cell of origin for aggressive breast cancer subtypes, suggest that TGFβ dysregulation during tumorigenesis may promote distinct breast cancer subtypes.