Current Molecular Medicine (v.14, #1)

Editorial A New Year of Excellence by David Wan-Cheng Li (1-2).

Hypothalamic mTOR: The Rookie Energy Sensor by P. B. Martínez de Morentin, N.. Martinez-Sanchez, J. Roa, J. Ferno, R. Nogueiras, M. Tena-Sempere, C. Dieguez, M. Lopez (3-21).
Optimal cellular function and therefore organism's survival is determined by the sensitive andaccurate convergence of energy and nutrient abundance to cell growth and division. Among other factors, thisintegration is coupled by the target of rapamycin (TOR) pathway, which is able to sense nutrient, energy andoxygen availability and also growth factor signaling. Indeed, TOR signaling regulates cell energy homeostasisby coordinating anabolic and catabolic processes for survival. TOR, named mTOR in mammals, is a conservedserine/threonine kinase that exists in two different complexes, mTORC1 and mTORC2. Recently, studies aresuggesting that alterations of those complexes promote disease and disrupted phenotypes, such as aging,obesity and related disorders and even cancer. The evidences linking mTOR to energy and metabolichomeostasis included the following. At central level mTOR regulates food intake and body weight beinginvolved in the mechanism by which signals such as leptin and ghrelin exert its effects. At peripheral level itinfluences adipogenesis and lipogenesis in different tissues including the liver. Noteworthy chronic nutritionalactivation of mTOR signaling has been implicated in the development of beta cell mass expansion and oninsulin resistance. Understanding of mTOR and other molecular switches, such as AMP-activated proteinkinase (AMPK), as well as their interrelationship is crucial to know how organisms maintain optimalhomeostasis. This review summarizes the role of hypothalamic TOR complex in cellular energy sensing,evidenced in the last years, focusing on the metabolic pathways where it is involved and the importance of thismetabolic sensor in cellular and whole body energy management. Understanding the exact role ofhypothalamic mTOR may provide new cues for therapeutic intervention in diseases.

The Protein-Protein Interaction-Mediated Inactivation of PTEN by J. De Melo, L. He, D. Tang (22-33).
PTEN (Phosphatase and Tensin homologue deleted on chromosome 10, 10q23.3) is the dominantphosphatase responsible for the dephosphorylation of the 3-position phosphate from the inositol ring ofphosphatidylinositol 3,4,5 triphosphate (PIP3), and thereby directly antagonizes the actions mediated byPhosphatidylinositol-3 Kinase (PI3K). PI3K functions in numerous pathways and cellular processes, includingtumourigenesis. Therefore, mechanisms regulating PTEN function, either positively or negatively are of greatinterest not only to oncogenesis but also to other aspects of human health. Since its discovery in 1997, PTENhas been one of the most-heavily studied tumour suppressors and has been the subject of numerous reviews.Most investigations and reviews center on PTEN's function and its regulation. While the regulation of PTENfunction via genetic and/or epigenetic mechanisms has been extensively studied, the impact of protein-proteininteraction on PTEN function remains less clear. Recent research has revealed that PTEN can be specificallyinhibited by its interaction with other proteins, which are collectively termed PTEN-negative regulators (PTENNRs).This review will summarize our current understanding on the protein network that influences PTENfunction with a specific focus on PTEN-NRs.

The Molecular Basis of Notch Signaling Regulation: A Complex Simplicity by R. Palermo, S. Checquolo, D. Bellavia, C. Talora, I. Screpanti (34-44).
The Notch receptors have attracted considerable attention for their ability to control cellular functionsthat regulate embryo development and tissue homeostasis. Notch receptors act by controlling the expressionof a specific set of target genes. If Notch signaling system can be so simple, and yet so complex in itspleiotropic effects, then a sophisticated network of regulatory mechanisms is required to maintain the controlover the initiation, activity and termination of this signaling pathway. A multitude of regulatory mechanisms hasbeen discovered that controls the interaction of Notch receptors with their ligands, the assembling of a Notchtranscriptional activation complex and the termination of Notch signals. The intracellular and extracellulardomains of the Notch receptors are synthesized as single proteins, pairing with each other during theirtrafficking through the exocytotic route. The mechanisms operating in the phase preceding the generation ofthe heterodimeric signal-competent Notch receptors can be as elaborate and physiologically important asthose operating downstream of Notch receptor activation. These regulatory mechanisms, which are essentialto understand the role of Notch signaling in human physiology and pathology are reviewed here.

The genetic alterations associated with breast carcinogenesis are well known. On the contraryepigenetic alterations in hereditary breast cancer are a new field. Two epigenetic mechanisms have emergedas the most critical players in transcriptional regulation in breast cancer: the methylation of DNA and microRNAinterference.;In this review we will focus on recent findings on gene silencing caused by DNA methylation and microRNA toexplore the potential role of these epigenetic changes in the understanding of hereditary breast cancer.Moreover we will describe the same alterations in basal-like breast cancer and in triple-negative breast cancer,since their phenotypes have similarities with BRCA1-mutated tumors. To underline the possibility that someepigenetic alterations could also be used as potential epigenetic biomarkers of drug sensitivity or resistance,we will discuss the more common therapies in hereditary breast cancer that could also be applied to breastcancer with basal-like or triple negative phenotypes.

New Advances in the Pathogenesis and Progression of Barrettµs Esophagus by M. M. Streppel, E. A. Montgomery, A. Maitra (58-68).
Barrett's esophagus (BE) is a premalignant condition in the esophagus, with a rising incidence rateamong Caucasians, and an established risk factor for the subsequent progression to esophagealadenocarcinoma (EAC). In contrast to the stratified squamous epithelium that normally lines the distalesophagus, BE is characterized by columnar epithelium that to some extent resembles the mucosa of thelower intestinal tract. The mechanism of intestinalization of the esophagus is still uncertain. For many years, itwas postulated that either abnormal differentiation of resident progenitor cells in the esophagus, ortransdifferentiation of mature esophageal keratinocytes provoked by reflux-induced genetic alterations,resulted in the BE phenotype. However, more recent studies suggest that indigenous progenitor cells at thegastro-esophageal junction might, under unfavorable conditions such as TP63 loss or an activatedinflammatory response, migrate to the esophagus and initiate columnar cell differentiation. In this review, wediscuss the competing theories of the origins of BE, as well as the role of developmental signaling pathwayssuch as Notch, Hedgehog, and Wnt/?-catenin signaling that have been implicated in the molecularpathogenesis of BE and EAC. Additionally, we provide an overview of the mutational landscapes of BE andEAC, derived from the results of recently published next generation sequencing (NGS) studies. Futureresearch should elucidate whether NGS on endoscopic mucosal biopsies can help in identifying BE patients athighest risk for EAC development, and whether some of the prevalent mutations are “actionable”, leading toimprovements in current therapeutic strategies for BE and EAC.

Molecular Diagnosis in Autoimmune Skin Blistering Conditions by J.V. Otten, T. Hashimoto, M. Hertl, A.S. Payne, C. Sitaru (69-95).
Blister formation in skin and mucous membranes results from a loss of cell-cell or cell-matrixadhesion and is a common outcome of pathological events in a variety of conditions, including autoimmuneand genetic diseases, viral and bacterial infections, or injury by physical and chemical factors. Autoantibodiesagainst structural components maintaining cell-cell and cell-matrix adhesion induce tissue damage inautoimmune blistering diseases. Detection of these autoantibodies either tissue-bound or circulating in serumis essential to diagnose the autoimmune nature of disease. Various immunofluorescence methods as well asmolecular immunoassays, including enzyme-linked immunosorbent assay and immunoblotting, belong to themodern diagnostic algorithms for these disorders. There is still a considerable need to increase awareness ofthe rare autoimmune blistering diseases, which often show a severe, chronic-relapsing course, amongphysicians and the public. This review article describes the immunopathological features of autoimmunebullous diseases and the molecular immunoassays currently available for their diagnosis and monitoring.

Nature has evolved DNA polymerases (Pols) with different replication fidelity with the purpose ofmaintaining and faithfully propagating the genetic information. Besides the four classical Pols (Pol ?, ?, ?, ?),mammalian cells contain at least twelve specialized Pols whose functions have been discovered recently andare still not completely elucidated. Among them, Pols belonging to the Y family contribute to cell survival bypromoting DNA damage tolerance. They are primarily involved in the translesion synthesis (TLS) pathway,incorporating dNTPs in an error-free or error-prone manner, depending on the nature of the DNA lesion. Froman evolutionary point of view, their high mutagenic potential seems to guarantee the proper flexibility of vitalimportance for both adaptation to a changeable environment and evolution of the species. These Pols aresubjected to a complex network of regulation, since their uncontrolled access to DNA might promotemutagenesis and neoplastic transformation. Altered expression of Y family is a hallmark of several tumortypes. In recent years, the unique structure and properties of Y family Pols have been exploited to designmolecules that selectively interfere with the Pol of interest with minimal effect on normal cells. In addition, theirdistinctive properties have been applied to innovative techniques, such as compartmentalized self-replication(CSR), short-patch CSR, phage display and molecular breeding. These approaches are based on mutant Polsprovided with novel and ameliorated features and find applications in various fields, from biotechnology todiagnostics, paleontology and forensic analysis.

Animal Mitochondria: Evolution, Function, and Disease by M. Tao, C.-P. You, R.-R. Zhao, S.-J. Liu, Z.-H. Zhang, C. Zhang, Y. Liu (115-124).
Mitochondria are sub-cellular organelles responsible for producing the majority of cellular energythrough the process of oxidative phosphorylation (OXPHOS), and are found in nearly all eukaryotic cells.Mitochondria have a unique genetic system, mitochondrial DNA (mtDNA), which is a small, self-replicating anddiverse genome. In the past 30 years, mtDNA has made significant contribution to molecular ecology andphylogeography. Mitochondria also represent a unique system of mitochondrial-nuclear genomic cooperation.Additionally, mitochondrial dysfunction can be fatal. In this paper, we review several aspects of mitochondria,including evolution and the origin of mitochondria, energy supply and the central role of mitochondria inapoptosis, and mitochondrial dysfunction. It is shown that mitochondria play a critical role in many aspects oflife.

Chemoprevention Gene Therapy (CGT) of Pancreatic Cancer Using Perillyl Alcohol and a Novel Chimeric Serotype Cancer Terminator Virus by S. Sarkar, B. Azab, B.A. Quinn, X. Shen, P. Dent, A.L. Klibanov, L. Emdad, S.K. Das, D. Sarkar, P.B. Fisher (125-140).
Conditionally replication competent adenoviruses (Ads) that selectively replicate in cancer cells andsimultaneously express a therapeutic cytokine, such as melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7), hold potential for treating humancancers. To enhance the efficacy of the CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiberbipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a Coxsackie Adenovirus receptor (CAR)independent manner, while retaining high infectivity in cancer cells containing high CAR. Although mda-7/IL-24displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells display anintrinsic resistance to mda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translational block. However,using a chemoprevention gene therapy (CGT) approach with perillyl alcohol (POH) and a replicationincompetent Ad to deliver mda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNA intoprotein resulting in pancreatic cancer cell death in vitro and in vivo in nude mice containing human PDACxenografts. This combination synergistically induces mda-7/IL-24-mediated cancer-specific apoptosis byinhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum (ER) stressresponse through induction of BiP/GRP-78, which is most evident in chimeric-modified non-replicating Ad.5/3-mda-7- and CTV-M7-infected PDAC cells. Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizestherapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 or Bcl-xL to mda-7/IL-24-mediatedapoptosis. Ad.5/3-CTV-M7 plus POH also exerts a significant antitumor 'bystander' effect in vivo suppressingboth primary and distant site tumor growth, confirming therapeutic utility of Ad.5/3-CTV-M7 plus POH in PDACtreatment, where all other current treatment strategies in clinical settings show minimal efficacy.

Topoisomerase I (topo I) is an essential nuclear enzyme involved in virtually all aspects of geneexpression, and is the target of the anti-cancer drugs- camptothecin (CPT) and its derivatives. Improvement ofthe survival rates of young women with cancer has led to the consideration of the effects of long-termchemotherapy on their fertility. The effect of anticancer drugs on ovarian function was previously investigated;however, no reports are available concerning their effect on the endometrium, whose integrity is an importantfactor in embryo implantation. Here we used a rat animal model to investigate the expression and activity oftopo I in the various physiologic phases of the endometrium and the influence of CPT on its integrity andreceptivity.;The results show, for the first time, that the endometrial topo I level and activity are influenced by thephysiologic phases of the endometrium (estrous cycle) and correlate with the estrogen blood concentration.Treatment with the anti-cancer drug CPT caused histological disruption of the endometrium and deleteriouseffect on its cyclicity. Moreover, CPT treatment significantly reduced the implantation rate of embryos,suggesting alteration in the receptivity of the endometrium.;These results suggest that topo I is important for maintaining the normal physiologic cyclicity and functionalityof the endometrium in rats. Anti-cancer agents that target topo I severely impair estrous cycle progression andendometrial integrity and receptivity, emphasizing the importance of addressing the effect of chemotherapy onthe endometrial functionality.

Id-1 is a member of the helix-loop-helix family of proteins that regulates the activity of transcriptionfactors to suppress cellular differentiation and to promote cell growth. Overexpression of Id-1 in tumor cellscorrelates with increased malignancy and resistance to chemotherapy and radiotherapy. Id-1B is an isoformgenerated by alternative splicing that differs from the classical Id-1 in the 13-C-terminal amino acids, whosefunction is at present unknown. We have studied the role of Id-1B in cancer and its expression inhealthy/malignant lung tissues. Overexpression of Id-1B in A549 lung and PC3 prostate cancer cells reducedanchorage-dependent and independent proliferation and clonogenic potential. Moreover, it increased theproportion of cells in the G0/G1 phase of the cell cycle and p27 levels, while reduced phospho-Erk and cyclin Alevels. Through microarray analysis, we identified genes involved in cell growth and proliferation that arespecifically deregulated as a consequence of Id-1B overexpression, including IGF2, BMP4, Id2, GATA3, EREGand AREG. Id-1B overexpressing cells that were treated with 4Gy irradiation dose were significantly lessresistant to cell death. In vivo assays demonstrated that tumors with high Id-1B levels exhibited less growth(p<0.01), metabolic activity (glucose uptake) and angiogenesis (p<0.05) compared to tumors with low Id-1Bexpression; mice survival was significantly extended (p<0.05). Quantification by qRT-PCR revealed thatexpression of Id-1B was significantly lower (p<0.01) in human lung tumors compared to their matched nonmalignantcounterparts. In conclusion, our results demonstrate that Id-1B decreases the malignancy of lungand prostate cancer cells, sensitizes them to radiotherapy-induced cell death, and counteracts the protumorigenicrole of the classical form of Id-1.

CD147 Promotes Melanoma Progression Through Hypoxia-Induced MMP2 Activation by W. Zeng, J. Su, L. Wu, D. Yang, T. Long, D. Li, Y. Kuang, J. Li, M. Qi, J. Zhang, X. Chen (163-173).
Hypoxia enhances MMP2 expression and the invasion and metastatic potential of melanoma cells.CD147 has been shown to induce MMP2 in multiple cancers. To investigate the role of CD147 in hypoxiainducedMMP2 activation, we performed immunohistochemistry (IHC) staining in 206 normal and melanomatissue samples, and analyzed the correlation between HIF1? and CD147. ChIP (chromosomeImmunoprecipitation) in melanoma cell lines supports that HIF1? directly binds to CD147 promoter. Moreover,we made a series of deletion mutants of CD147 promoter, and identified a conserved HIF1? binding site. Pointmutation in this site significantly decreased CD147 response to hypoxia. Importantly, knocking down CD147attenuates MMP2 response to hypoxia in melanoma cell lines. MMP2 could not be efficiently activated byhypoxia in CD147 depletion cells. ELISA data showed that MMP2 secretion was reduced in CD147 depletioncells than control under hypoxia condition. To verify the data from cell culture model, we performed in vivomouse xenograft experiment. IHC staining showed reduced MMP2 level in CD147 depleted xenograftscompared to the control group, with the HIF1? level being comparable. Our study demonstrates a novelpathway mediated by CD147 to promote the MMP2 activation induced by hypoxia, and helps to understand theinterplay between hypoxia and melanoma progression.

Aberrant Expression of CXCR4 Significantly Contributes to Metastasis and Predicts Poor Clinical Outcome in Breast Cancer by P. Yang, S.-X. Liang, W.-H. Huang, H.-W. Zhang, X.-L. Li, L.-H. Xie, C.-W. Du, G.-J. Zhang (174-184).
Triple negative breast cancer is known for its visceral metastasis. We have found that CXCR4 isoverexpressed in triple negative breast cancer and is associated with visceral metastasis. We furtherinvestigated whether CXCR4 is a prognostic factor affecting survival following visceral metastasis in breastcancer patients. Our results indicate that increased CXCR4 expression among breast cancer patients with visceralmetastasis was positively correlated with poor overall survival (P<0.001). Silencing of CXCR4 was associatedwith a decrease in the tumorigenic properties of MDA-MB-231 breast cancer cells, caused reversion of EMT andsuppression of MMP-9, increased apoptosis, and caused a reduced incidence of tumor lung metastasis in mice.These results are indicative of CXCR4 having a predictive role in patients with visceral metastasis and indicatethat shRNA knock down of CXCR4 might be a novel therapeutic strategy to prevent breast cancer metastasiswhen CXCR4 is overexpressed.

Glioblastoma is highly resistant to radiation therapy. The underlying molecular mechanism is notcompletely understood. The DNA damage response (DDR) pathway plays a crucial role in radioresistance ofglioablastoma cells. Growing evidence has demonstrated that radiation induces alterations in microRNA (miR)profiles. However, how radiation induces specific miRs and how they might regulate the DDR remain elusive.In our study, we found that radiation induced c-jun transcription of miR-221 and miR-222. miR-221 and miR-222 modulated DNA-PKcs expression to affect DNA damage repair by activating Akt independent of PTENstatus. Knocking down of miR-221/222 significantly increased radiosensitivity of glioblastoma cells. Inhibition ofAkt by RNAi or LY294002 treatment may overcome miR-221/222 induced radioresistance. Notably, combinedanti-miR-221/222 and radiotherapy has remarkably inhibited tumor growth compared with anti-miR-221/222 orradiotherapy alone in a subcutaneous mouse model. Our results suggest that radio-induced c-jun promotestranscription of miR-221/222, which mediates DNA damage repair of glioblastoma cells independent of PTEN.These data indicate for the first time that miR-221/222 play an important role in mediating radio-induced DNAdamage repair and that miR-221/222 could serve as potential therapeutic targets for increasing radiosensitivityof glioblastoma cells.