Rational design of small molecule RHOA inhibitors for gastric cancer.

Previously, we identified Ras homologous A (RHOA) as a major signaling hub in gastric cancer (GC), the third most common cause of cancer death in the world, prompting us to rationally design an efficacious inhibitor of this oncogenic GTPase. Here, based on that previous work, we extend those computational analyses to further pharmacologically optimize anti-RHOA hydrazide derivatives for greater anti-GC potency. Two of these, JK-136 and JK-139, potently inhibited cell viability and migration/invasion of GC cell lines, and mouse xenografts, diversely expressing RHOA. Moreover, JK-136's binding affinity for RHOA was >140-fold greater than Rhosin, a nonclinical RHOA inhibitor. Network analysis of JK-136/-139 vs. Rhosin treatments indicated downregulation of the sphingosine-1-phosphate, as an emerging cancer metabolic pathway in cell migration and motility. We assert that identifying and targeting oncogenic signaling hubs, such as RHOA, represents an emerging strategy for the design, characterization, and translation of new antineoplastics, against gastric and other cancers.

Lipopolysaccharide treatment induces genome-wide pre-mRNA splicing pattern changes in mouse bone marrow stromal stem cells.

BACKGROUND: Lipopolysaccharide (LPS) is a gram-negative bacterial antigen that triggers a series of cellular responses. LPS pre-conditioning was previously shown to improve the therapeutic efficacy of bone marrow stromal cells/bone-marrow derived mesenchymal stem cells (BMSCs) for repairing ischemic, injured tissue. RESULTS: In this study, we systematically evaluated the effects of LPS treatment on genome-wide splicing pattern changes in mouse BMSCs by comparing transcriptome sequencing data from control vs. LPS-treated samples, revealing 197 exons whose BMSC splicing patterns were altered by LPS. Functional analysis of these alternatively spliced genes demonstrated significant enrichment of phosphoproteins, zinc finger proteins, and proteins undergoing acetylation. Additional bioinformatics analysis strongly suggest that LPS-induced alternatively spliced exons could have major effects on protein functions by disrupting key protein functional domains, protein-protein interactions, and post-translational modifications. CONCLUSION: Although it is still to be determined whether such proteome modifications improve BMSC therapeutic efficacy, our comprehensive splicing characterizations provide greater understanding of the intracellular mechanisms that underlie the therapeutic potential of BMSCs.

Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.

BACKGROUND: "Biomarker-driven targeted therapy," the practice of tailoring patients' treatment to the expression/activity levels of disease-specific genes/proteins, remains challenging. For example, while the anti-ERBB2 monoclonal antibody, trastuzumab, was first developed using well-characterized, diverse in vitro breast cancer models (and is now a standard adjuvant therapy for ERBB2-positive breast cancer patients), trastuzumab approval for ERBB2-positive gastric cancer was largely based on preclinical studies of a single cell line, NCI-N87. Ensuing clinical trials revealed only modest patient efficacy, and many ERBB2-positive gastric cancer (GC) patients failed to respond at all (i.e., were inherently recalcitrant), or succumbed to acquired resistance. METHOD: To assess mechanisms underlying GC insensitivity to ERBB2 therapies, we established a diverse panel of GC cells, differing in ERBB2 expression levels, for comprehensive in vitro and in vivo characterization. For higher throughput assays of ERBB2 DNA and protein levels, we compared the concordance of various laboratory quantification methods, including those of in vitro and in vivo genetic anomalies (FISH and SISH) and xenograft protein expression (Western blot vs. IHC), of both cell and xenograft (tissue-sectioned) microarrays. RESULTS: The biomarker assessment methods strongly agreed, as did correlation between RNA and protein expression. However, although ERBB2 genomic anomalies showed good in vitro vs. in vivo correlation, we observed striking differences in protein expression between cultured cells and mouse xenografts (even within the same GC cell type). Via our unique pathway analysis, we delineated a signaling network, in addition to specific pathways/biological processes, emanating from the ERBB2 signaling cascade, as a potential useful target of clinical treatment. Integrated analysis of public data from gastric tumors revealed frequent (10 - 20 %) amplification of the genes NFKBIE, PTK2, and PIK3CA, each of which resides in an ERBB2-derived subpathway network. CONCLUSION: Our comprehensive bioinformatics analyses of highly heterogeneous cancer cells, combined with tumor "omics" profiles, can optimally characterize the expression patterns and activity of specific tumor biomarkers. Subsequent in vitro and in vivo validation, of specific disease biomarkers (using multiple methodologies), can improve prediction of patient stratification according to drug response or nonresponse.

Natural Polyphenols in Cancer Chemoresistance.

Resistance to chemotherapy remains a major impediment to the management of most types of cancer. Both intrinsic and acquired drug resistance are mediated by several cellular and molecular mechanisms, including alternative growth-signaling pathways unaffected by specific therapies, alterations in the tumor microenvironment (e.g., hypoxia and angiogenesis), and active transport of drugs out of the cell. Epidemiological studies have validated an inverse correlation between the consumption of dietary polyphenols and the risk of cancer, which has been attributed to polyphenol antioxidant capacity and their potential to inhibit activation of procarcinogens, cancer cell proliferation, metastasis, and angiogenesis, and inhibition or downregulation of active drug efflux transporters. Moreover, polyphenols can induce apoptosis in cancer cells and modulate immune responses and inflammatory cascades. Augmentation of the efficacy of chemotherapy and prevention of multidrug resistance are other important effects of dietary polyphenols that deserve further research, especially after the discovery of tight "crosstalk" between aberrant growth signaling and metabolic dysfunction in cancer cells. In this review, we cover what is currently known about the role of natural polyphenolic compounds in overcoming cancer drug resistance mediated by diverse primary and secondary resistance mechanisms.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

Epigenetic targeting therapies to overcome chemotherapy resistance.

It is now well established that epigenetic aberrations occur early in malignant transformation, raising the possibility of identifying chemopreventive compounds or reliable diagnostic screening using epigenetic biomarkers. Combinatorial therapies effective for the reexpression of tumor suppressors, facilitating resensitization to conventional chemotherapies, hold great promise for the future therapy of cancer. This approach may also perturb cancer stem cells and thus represent an effective means for managing a number of solid tumors. We believe that in the near future, anticancer drug regimens will routinely include epigenetic therapies, possibly in conjunction with inhibitors of "stemness" signal pathways, to effectively reduce the devastating occurrence of cancer chemotherapy resistance.

A Narrative Review of Alternative Symptomatic Treatments for Herpes Simplex Virus.

Herpes simplex virus-1 (HSV-1) and -2 (HSV-2) are large, spherically shaped, double-stranded DNA viruses that coevolved with Homo sapiens for over 300,000 years, having developed numerous immunoevasive mechanisms to survive the lifetime of their human host. Although in the continued absence of an acceptable prophylactic and therapeutic vaccine, approved pharmacologics (e.g., nucleoside analogs) hold benefit against viral outbreaks, while resistance and toxicity limit their universal application. Against these shortcomings, there is a long history of proven and unproven home remedies. With the breadth of purported alternative therapies, patients are exposed to risk of harm without proper information. Here, we examined the shortcomings of the current gold standard HSV therapy, acyclovir, and described several natural products that demonstrated promise in controlling HSV infection, including lemon balm, lysine, propolis, vitamin E, and zinc, while arginine, cannabis, and many other recreational drugs are detrimental. Based on this literature, we offered recommendations regarding the use of such natural products and their further investigation.

Empirical bayes model comparisons for differential methylation analysis.

A number of empirical Bayes models (each with different statistical distribution assumptions) have now been developed to analyze differential DNA methylation using high-density oligonucleotide tiling arrays. However, it remains unclear which model performs best. For example, for analysis of differentially methylated regions for conservative and functional sequence characteristics (e.g., enrichment of transcription factor-binding sites (TFBSs)), the sensitivity of such analyses, using various empirical Bayes models, remains unclear. In this paper, five empirical Bayes models were constructed, based on either a gamma distribution or a log-normal distribution, for the identification of differential methylated loci and their cell division-(1, 3, and 5) and drug-treatment-(cisplatin) dependent methylation patterns. While differential methylation patterns generated by log-normal models were enriched with numerous TFBSs, we observed almost no TFBS-enriched sequences using gamma assumption models. Statistical and biological results suggest log-normal, rather than gamma, empirical Bayes model distribution to be a highly accurate and precise method for differential methylation microarray analysis. In addition, we presented one of the log-normal models for differential methylation analysis and tested its reproducibility by simulation study. We believe this research to be the first extensive comparison of statistical modeling for the analysis of differential DNA methylation, an important biological phenomenon that precisely regulates gene transcription.

Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications.

Unlike epigenetic silencing of tumor suppressor genes, the role of epigenetic derepression of cancer-promoting genes or oncogenes in carcinogenesis remains less well understood. The tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer and their overexpression was previously reported to promote the migration and invasion of ovarian epithelial cells. Here, we show that the expression of claudin-3 and claudin-4 is repressed in ovarian epithelial cells in association with promoter 'bivalent' histone modifications, containing both the activating trimethylated histone H3 lysine 4 (H3K4me3) mark and the repressive mark of trimethylated histone H3 lysine 27 (H3K27me3). During ovarian tumorigenesis, derepression of CLDN3 and CLDN4 expression correlates with loss of H3K27me3 in addition to trimethylated histone H4 lysine 20 (H4K20me3), another repressive histone modification. Although CLDN4 repression was accompanied by both DNA hypermethylation and repressive histone modifications, DNA methylation was not required for CLDN3 repression in immortalized ovarian epithelial cells. Moreover, activation of both CLDN3 and CLDN4 in ovarian cancer cells was associated with simultaneous changes in multiple histone modifications, whereas H3K27me3 loss alone was insufficient for their derepression. CLDN4 repression was robustly reversed by combined treatment targeting both DNA demethylation and histone acetylation. Our study strongly suggests that in addition to the well-known chromatin-associated silencing of tumor suppressor genes, epigenetic derepression by the conversely related loss of repressive chromatin modifications also contributes to ovarian tumorigenesis via activation of cancer-promoting genes or candidate oncogenes.

Computational analysis of microRNA profiles and their target genes suggests significant involvement in breast cancer antiestrogen resistance.

MOTIVATION: Recent evidence shows significant involvement of microRNAs (miRNAs) in the initiation and progression of numerous cancers; however, the role of these in tumor drug resistance remains unknown. RESULTS: By comparing global miRNA and mRNA expression patterns, we examined the role of miRNAs in resistance to the 'pure antiestrogen' fulvestrant, using fulvestrant-resistant MCF7-FR cells and their drug-sensitive parental estrogen receptor (ER)-positive MCF7 cells. We identified 14 miRNAs downregulated in MCF7-FR cells and then used both TargetScan and PITA to predict potential target genes. We found a negative correlation between expression of these miRNAs and their predicted target mRNA transcripts. In genes regulated by multiple miRNAs or having multiple miRNA-targeting sites, an even stronger negative correlation was found. Pathway analyses predicted these miRNAs to regulate specific cancer-associated signal cascades. These results suggest a significant role for miRNA-regulated gene expression in the onset of breast cancer antiestrogen resistance, and an improved understanding of this phenomenon could lead to better therapies for this often fatal condition.

Enriched transcription factor binding sites in hypermethylated gene promoters in drug resistant cancer cells.

MOTIVATION: In the human genome, 'CpG islands', CG-rich regions located in or near gene promoters, are normally unmethylated. However, in cancer cells, CpG islands frequently gain methylation, resulting in silencing of growth-limiting tumor suppressor genes. To our knowledge, the potential relationship between CpG island hypermethylation, transcription factor (TF) binding in local promoter regions and transcriptional control has not been previously explored in a genome-wide context. RESULTS: In this study, we utilized bioinformatics tools and TF binding site(TFBs) databases to globally analyze sequences methylated in a laboratory model for the development of drug-resistant cancer. Our results demonstrated that four TFBS were enriched in hypermethylated sequences. More interestingly, overrepresentation of these TFBS was observed in hyper-/hypo-methylated sequences where signi.cant changes in methylation levels were observed in drug-resistant cancer cells. In summary, we believe that these.ndings offer a means to further explore the relationship between DNA methylation and gene expression in drug resistance and tumorigenesis.

Methyl group acceptance assay for the determination of global DNA methylation levels.

DNA methylation levels are affected by numerous environmental influences, including diet and xenobiotic exposure, and neoplasia has been firmly associated with genomic hypomethylation and localized hypermethylation of tumor suppressor genes. To reverse methylation-induced gene repression, DNA hypomethylating agents are currently in clinical trials for various malignancies, with two of these now approved for the therapy of myelodysplastic syndrome, and the efficacy of these drugs can be assessed by the monitoring of global DNA methylation levels. Herein, we outline a simple, well-established method for the evaluation of genomic DNA methylation levels, based on the ability of isolated DNA to "accept" radiolabeled methyl groups from S-[3H-methyl] adenosylmethionine, using the bacterial CpG methyltransferase SssI. As this enzyme methylates all unmethylated CpG dinucleotides in the genome, radiolabeled methyl group acceptance is inversely proportional to the level of preexisting methylation. This assay is applicable to a number of translational and basic research questions.

Systematic Inspection of the Clinical Relevance of TP53 Missense Mutations in Gastric Cancer.

The "guardian of the genome," TP53, is one of the most frequently mutated genes of all cancers. Despite the important biological roles of TP53, the clinical relevance of TP53 mutations, in gastric cancer (GC), remains largely unknown. Here, we systematically assessed clinical relevance, in terms of TP53 mutation positions, finding substantial variability. Thus, we hypothesized that the position of the TP53 mutation might affect clinical outcomes in GC. We systematically inspected missense mutations in TP53, from a TCGA (The Cancer Genome Atlas) GC dataset in UCSC Xena repository. Specifically, we examined five aspects of each mutational position: (1) the whole gene body; (2) known hot-spots; (3) the DNA-binding domain; (4) the secondary structure of the domain; and (5) individual mutation positions. We then analyzed the clinical outcomes for each aspect. These results showed that, in terms of secondary structure, patients with mutations in turn regions showed poor prognosis, compared to those with mutations in beta strand regions (log rank ${\text{p}}= {{0.043}}$p=0.043). Also, in terms of individual mutation positions, patients having mutations at R248 showed poorer survival than other patients having mutations at different TP53 positions (log rank ${\text{p}}= {{0.035}}$p=0.035).

TET1 promotes 5hmC-dependent stemness, and inhibits a 5hmC-independent epithelial-mesenchymal transition, in cervical precancerous lesions.

DNA hypermethylation is a driving force in carcinogenesis. However, the role of active DNA hypomethylation in cancer remains largely unknown. This process, facilitated by ten-eleven translocation methylcytosine dioxygenase 1 (TET1), which oxidizes 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), has never been studied in cervical cancer. Here, we found that TET1 and 5hmC correlative increases from normal cervix to Low-grade squamous intraepithelial lesion (LSIL), maximizing in High-grade squamous intraepithelial lesion (HSIL), and decreasing in invasive cancer. Full-length HPV-immortalized HSIL cells demonstrated higher TET1/5hmC levels, and stemness properties, compared to invasive cancer cells. TET1 silencing promoted the epithelial-mesenchymal transition (EMT), to transform precancerous cells in vivo. TET1 increased 5hmC in the ZEB1 and VIM promoters, surprisingly, silencing both genes. TET1 interaction with the histone modifiers, LSD1 and EZH2, on the ZEB1 promoter, resulted in gene silencing, via loss of histone H3K4 trimethylation, and gain of histone H3K27 trimethylation. Taken together, TET1 promotes stemness properties, and inhibits EMT, in HSIL cells, through 5hmC-dependent and -independent mechanisms.

The Epigenomics of Embryonic Pathway Signaling in Colorectal Cancer.

Colorectal cancer (CRC) is the second-leading cause of cancer death in developed countries. While early detection (e.g., colonoscopy) generally yields excellent outcomes, metastatic and drug-resistant disease is uniformly fatal, and non-compliance for screening remains over 25%. Familial CRCs (10% of total cases) primarily include mutations in the gene APC. Somatic disease is linked to several environmental several risk factors, including mutations in WNT, KRAS, and TGFß. To reflect the genesis/progression of CRC, a series of five discrete stages, from normal colon mucosa to fully invasive carcinoma, each regulated by specific "gatekeeper" genes, remains well-accepted after 20 years. However, many CRC tumors do not possess those particular mutations, suggesting alternative mechanisms. More recently, embryo-like "cancer stem cells" have been proposed to undergo self-renewal and drive tumorigenesis (and possibly, metastasis), as governed by specific "epigenomic" alterations. Here, we review recent literature describing possible mechanisms that underlie these phenotypes, including cancer "stemness," believed by many to associate with the epithelial-to-mesenchymal transition (EMT). We further propose that the maintenance of undifferentiated phenotypes, by the activity of distinct transcription factors, facilitates chromatin remodeling and phenotypic plasticity. With that regard, we support recent assertions that EMT is not an "either/or" event, but rather a continuous spectrum of mesenchymal vs. epithelial phenotypes (in various degrees of aberrant differentiation/undifferentiation). Finally, we discuss possible methods of pharmacologically targeting such aberrant epigenomes, with regard to their possible relevance toward halting, or even reversing, colorectal cancer progression.

Tackling multidrug resistance mediated by efflux transporters in tumor-initiating cells.

INTRODUCTION: Expression of the multifunctional ATP-binding cassette (ABC) efflux transporter gene family is a well-established mechanism for protecting cancer stem cells (CSCs) from damage or death due to toxins. The outcome of such protection makes CSCs innately multidrug resistant (MDR) to conventional chemotherapy. AREAS COVERED: While research has focused on gaining better insight into the role of ABC transporters in CSC drug resistance, various strategies to circumvent the function of these transporters have been proposed, including inhibition of transporter function through targeted tyrosine kinase inhibitors, competitive and allosteric modulators, shRNA-mediated inhibition, nanoparticle-mediated delivery of inhibitors, and modulating the regulation of transcriptional and signaling pathways involving ABC transporters. This review highlights the role of MDR mediated by ABC transporters, particularly in CSCs, and the current progress and pitfalls of strategies to circumvent MDR in CSCs. EXPERT OPINION: Cancer stem cells are now a subject of intense research, as it is hypothesized that these progenitors predominantly beget tumorigenesis, chemoresistance, and metastasis. Consequently, the design and synthesis of more effective ABC transporter inhibitors, to increase cytotoxic drug concentrations in CSCs (thus increasing their eradication), is a promising approach for the field of oncology.

Pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolines: Novel compounds that reverse ABCG2-mediated resistance in cancer cells.

Overexpression of ATP-binding cassette transporter (ABC) subfamily G2 in cancer cells is known to elicit a MDR phenotype, ultimately resulting in cancer chemotherapy failure. Here, we report, for the first time, the effect of eight novel pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline (IND) derivatives that inhibit ABCG2 transporter restoring cancer cell chemosensitivity. IND -4, -5, -6, -7, and -8, at 10 µM, and nilotinib at 5 µM, significantly potentiated (8-10 fold) the cytotoxicity of the ABCG2 substrates mitoxantrone (MX) and doxorubicin in HEK293 cells overexpressing ABCG2 transporter, MX (~14 fold) in MX-resistant NCI-H460/MX-20 small cell lung cancer, and of topotecan (~7 fold) in S1-M1-80 colon cancer cells which all stably expressing ABCG2. In contrast, cytotoxicity of cisplatin, which is not an ABCG2 substrate, was not altered. IND-5,-6,-7, and -8 significantly increased the accumulation of rhodamine-123 in multidrug resistant NCI-H460/MX-20 cells overexpressing ABCG2. Both IND-7 and -8, the most potent ABCG2 inhibitors, had the highest affinities for the binding sites of ABCG2 in modeling studies. In conclusion, the beneficial actions of new class of agents warrant further development as potential MDR reversal agents for clinical anticancer agents that suffer from ABCG2-mediated MDR insensitivity.

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

Network Comparison of Inflammation in Colorectal Cancer and Alzheimer's Disease.

Recently, a large clinical study revealed an inverse correlation of individual risk of cancer versus Alzheimer's disease (AD). However, no explanation exists for this anticorrelation at the molecular level; however, inflammation is crucial to the pathogenesis of both diseases, necessitating a need to understand differing signaling usage during inflammatory responses distinct to both diseases. Using a subpathway analysis approach, we identified numerous well-known and previously unknown pathways enriched in datasets from both diseases. Here, we present the quantitative importance of the inflammatory response in the two disease pathologies and summarize signal transduction pathways common to both diseases that are affected by inflammation.