Alexander Yu. Nikitin

Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation

Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimers disease and cancer.

MicroRNA-34b and MicroRNA-34c Are Targets of p53 and Cooperate in Control of Cell Proliferation and Adhesion-Independent Growth

MicroRNAs (miRNA) are a recently discovered class of noncoding RNAs that negatively regulate gene expression. Recent evidence indicates that miRNAs may play an important role in cancer. However, the mechanism of their deregulation in neoplastic transformation has only begun to be understood. To elucidate the role of tumor suppressor p53 in regulation of miRNAs, we have analyzed changes in miRNA microarray expression profile immediately after conditional inactivation of p53 in primary mouse ovarian surface epithelium cells. Among the most significantly affected miRNAs were miR-34b and miR-34c, which were down-regulated 12-fold according to quantitative reverse transcription-PCR analysis. Computational promoter analysis of the mir-34b/mir-34c locus identified the presence of evolutionarily conserved p53 binding sites approximately 3 kb upstream of the miRNA coding sequence. Consistent with evolutionary conservation, mir-34b/mir-34c were also down-regulated in p53-null human ovarian carcinoma cells. Furthermore, as expected from p53 binding to the mir-34b/c promoter, doxorubicin treatment of wild-type, but not p53-deficient, cells resulted in an increase of mir-34b/mir-34c expression. Importantly, miR-34b and miR-34c cooperate in suppressing proliferation and soft-agar colony formation of neoplastic epithelial ovarian cells, in agreement with the partially overlapping spectrum of their predicted targets. Taken together, these results show the existence of a novel mechanism by which p53 suppresses such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation.

Classification of Proliferative Pulmonary Lesions of the Mouse Recommendations of the Mouse Models of Human Cancers Consortium

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Frequent Downregulation of miR-34 Family in Human Ovarian Cancers

Purpose: The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. Experimental Designs: miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. Results: miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Conclusions: Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC. Clin Cancer Res; 16(4); 1119–28

Synergy of p53 and Rb Deficiency in a Conditional Mouse Model for Metastatic Prostate Cancer

Pathways mediated by p53 and Rb are frequently altered in aggressive human cancers, including prostate carcinoma. To test directly the roles of p53 and Rb in prostate carcinogenesis, we have conditionally inactivated these genes in the prostate epithelium of the mouse. Inactivation of either p53 or Rb leads to prostatic intraepithelial neoplasia developing from the luminal epithelium by 600 days of age. In contrast, inactivation of both genes results in rapidly developing (median survival, 226 days) carcinomas showing both luminal epithelial and neuroendocrine differentiation. The resulting neoplasms are highly metastatic, resistant to androgen depletion from the early stage of development, and marked with multiple gene expression signatures commonly found in human prostate carcinomas. Interestingly, gains at 4qC3 and 4qD2.2 and loss at 14qA2-qD2 have been consistently found by comparative genomic hybridization. These loci contain such human cancer-related genes as Nfib, L-myc, and Nkx3.1, respectively. Our studies show a critical role for p53 and Rb deficiency in prostate carcinogenesis and identify likely secondary genetic alterations. The new genetically defined model should be particularly valuable for providing new molecular insights into the pathogenesis of human prostate cancer.

Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer deaths among women in the United States, but its pathogenesis is poorly understood. Some epithelial cancers are known to occur in transitional zones between two types of epithelium, whereas others have been shown to originate in epithelial tissue stem cells. The stem cell niche of the ovarian surface epithelium (OSE), which is ruptured and regenerates during ovulation, has not yet been defined unequivocally. Here we identify the hilum region of the mouse ovary, the transitional (or junction) area between the OSE, mesothelium and tubal (oviductal) epithelium, as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are cycling slowly and express stem and/or progenitor cell markers ALDH1, LGR5, LEF1, CD133 and CK6B. These cells display long-term stem cell properties ex vivo and in vivo, as shown by our serial sphere generation and long-term lineage-tracing assays. Importantly, the hilum cells show increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1, whose pathways are altered frequently in the most aggressive and common type of human EOC, high-grade serous adenocarcinoma. Our study supports experimentally the idea that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis.

c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart

We examined the myogenic response to infarction in neonatal and adult mice to determine the role of c-kit+ cardiovascular precursor cells (CPC) that are known to be present in early heart development. Infarction of postnatal day 1–3 c-kitBAC-EGFP mouse hearts induced the localized expansion of (c-kit)EGFP+ cells within the infarct, expression of the c-kit and Nkx2.5 mRNA, myogenesis, and partial regeneration of the infarction, with (c-kit)EGFP+ cells adopting myogenic and vascular fates. Conversely, infarction of adult mice resulted in a modest induction of (c-kit)EGFP+ cells within the infarct, which did not express Nkx2.5 or undergo myogenic differentiation, but adopted a vascular fate within the infarction, indicating a lack of authentic CPC. Explantation of infarcted neonatal and adult heart tissue to scid mice, and adoptive transfer of labeled bone marrow, confirmed the cardiac source of myogenic (neonate) and angiogenic (neonate and adult) cells. FACS-purified (c-kit)EGFP+/(αMHC)mCherry− (noncardiac) cells from microdissected infarcts within 6 h of infarction underwent cardiac differentiation, forming spontaneously beating myocytes in vitro; cre/LoxP fate mapping identified a noncardiac population of (c-kit)EGFP+ myocytes within infarctions, indicating that the induction of undifferentiated precursors contributes to localized myogenesis. Thus, adult postinfarct myogenic failure is likely not due to a context-dependent restriction of precursor differentiation, and c-kit induction following injury of the adult heart does not define precursor status.

Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARβ/δ to RAR

Retinoic acid (RA) displays potent anticarcinogenic activities that are mediated by the nuclear retinoic acid receptors (RARs). However, use of RA in oncology is limited by RA resistance acquired during carcinogenesis. Moreover, in some cancers, RA facilitates rather than inhibits growth. A clue to this paradoxical behavior was recently suggested by the findings that RA also activates PPARβ/δ, a receptor involved in mitogenic and anti-apoptotic activities. The observations that partitioning of RA between its two receptors is regulated by two intracellular lipid-binding proteins—CRABP-II, which targets RA to RAR, and FABP5, which delivers it to PPARβ/δ—further suggest that RA resistance may stem from the deregulation of the binding proteins, resulting in activation of PPARβ/δ rather than RAR. Here, we show that, in the RA-resistant mouse model of breast cancer MMTV-neu, RA indeed activates the nonclassical RA receptor PPARβ/δ. This behavior was traced to an aberrantly high intratumor FABP5/CRABP-II ratio. Decreasing this ratio in mammary tissue diverted RA from PPARβ/δ to RAR and suppressed tumor growth. The data demonstrate the existence of a mechanism that underlies RA resistance in tumors, indicate that CRABP-II functions as a tumor suppressor, and suggest that the inhibition of FABP5 may comprise a therapeutic strategy for overcoming RA resistance in some tumors.

Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis.

The mammalian circadian system has been implicated in the regulation of various biological processes including those involved in genotoxic stress responses and tumor suppression. Here we report that mice with the functional deficiency in circadian transcription factor CLOCK (Clock/Clock mutant mice) do not display predisposition to tumor formation both during their normal lifespan or when challenged by γ-radiation. This phenotype is consistent with high apoptotic and low proliferation rate in lymphoid tissues of Clock mutant mice and is supported by the gene expression profiling of a number of apoptosis and cell cycle-related genes, as well as by growth inhibition of cells with CLOCK downregulation. At the same time, Clock mutant mice respond to low-dose irradiation by accelerating their aging program, and develop phenotypes that are reminiscent of those in Bmal1-deficient mice. Taken together, our results demonstrate the dichotomy in biological consequences of the disruption of the circadian clock with respect to ageing and cancer. They also highlight the existence of a complex interconnection between ageing, carcinogenesis and individual components of the circadian clock machinery.

Somatic Mutation of p53 Leads to Estrogen Receptor -Positive and -Negative Mouse Mammary Tumors with High Frequency of Metastasis

Approximately 70% of human breast cancers are estrogen receptor α (ERα)-positive, but the origins of ERα-positive and -negative tumors remain unclear. Hormonal regulation of mammary gland development in mice is similar to that in humans; however, most mouse models produce only ERα-negative tumors. In addition, these mouse tumors metastasize at a low rate relative to human breast tumors. We report here that somatic mutations of p53 in mouse mammary epithelial cells using the Cre/loxP system leads to ERα-positive and -negative tumors. p53 inactivation under a constitutive active WAPCrec in prepubertal/pubertal mice, but not under MMTVCre in adult mice, leads to the development of ERα-positive tumors, suggesting that target cells or developmental stages can determine ERα status in mammary tumors. Importantly, these tumors have a high rate of metastasis. An inverse relationship between the number of targeted cells and median tumor latency was also observed. Median tumor latency reaches a plateau when targeted cell numbers exceed 20%, implying the existence of saturation kinetics for breast carcinogenesis. Genetic alterations commonly observed in human breast cancer including c-myc amplification and Her2/Neu/erbB2 activation were seen in these mouse tumors. Thus, this tumor system reproduces many important features of human breast cancer and provides tools for the study of the origins of ERα-positive and -negative breast tumors in mice.