Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

Cancer is a multi-step process that can be viewed as a cellular and immunological shift away from homeostasis in response to selected infectious agents, mutations, diet, and environmental carcinogens. Homeostasis, which contributes importantly to the definition of “health,” is maintained, in part by the production of short-chain fatty acids (SCFAs), which are metabolites of specific gut bacteria. Alteration in the composition of gut bacteria, or dysbiosis, is often a major risk factor for some two dozen tumor types. Dysbiosis is often characterized by diminished levels of SCFAs in the stool, and the presence of a “leaky gut,” permitting the penetration of microbes and microbial derived molecules (e.g., lipopolysaccharides) through the gut wall, thereby triggering chronic inflammation. SCFAs attenuate inflammation by inhibiting the activation of nuclear factor kappa B, by decreasing the expression of pro-inflammatory cytokines such as tumor necrosis factor alpha, by stimulating the expression of anti-inflammatory cytokines such as interleukin-10 and transforming growth factor beta, and by promoting the differentiation of naïve T cells into T regulatory cells, which down-regulate immune responses by immunomodulation. SCFA function epigenetically by inhibiting selected histone acetyltransferases that alter the expression of multiple genes and the activity of many signaling pathways (e.g., Wnt, Hedgehog, Hippo, and Notch) that contribute to the pathogenesis of cancer. SCFAs block cancer stem cell proliferation, thereby potentially delaying or inhibiting cancer development or relapse by targeting genes and pathways that are mutated in tumors (e.g., epidermal growth factor receptor, hepatocyte growth factor, and MET) and by promoting the expression of tumor suppressors (e.g., by up-regulating PTEN and p53). When administered properly, SCFAs have many advantages compared to probiotic bacteria and fecal transplants. In carcinogenesis, SCFAs are toxic against tumor cells but not to surrounding tissue due to differences in their metabolic fate. Multiple hallmarks of cancer are also targets of SCFAs. These data suggest that SCFAs may re-establish homeostasis without overt toxicity and either delay or prevent the development of various tumor types.

Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro , GLS4 was significantly less toxic for primary human hepatocytes ( P < 0.01 up to 100 μM), inhibited virus accumulation in the supernantant of HepAD38 cells ( P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC ₅₀ ) ( P < 0.02), and more strongly inhibited core gene expression ( P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

Hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. HBx transcriptionally up-regulates the expression of a unique gene, URG11, which in turn transcriptionally up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. HBx and URG11 also alter the expression of multiple microRNAs, and by miRNA array analysis, both were shown to promote the expression of miR-148a. Elevated miR-148a was also seen in HBx positive liver samples from infected patients. To study the function of miR-148a, anti-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Introduction of anti-miR-148a increased PTEN protein and mRNA expression, suggesting that PTEN was targeted by miR-148a. Anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants in the 3′UTR of PTEN mRNA. Introduction of anti-miR-148a also resulted in depressed Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin. Thus, miR-148a may play a central role in HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The HBV encoded oncoprotein, HBx, alters the expression of host genes and the activity of multiple signal transduction pathways that contribute to the pathogenesis of HCC by multiple mechanisms independent of HBV replication. However, it is not clear which pathways are the most relevant therapeutic targets in hepatocarcinogenesis. Short chain fatty acids (SCFAs) have strong anti-inflammatory and anti-neoplastic properties, suggesting that they may block the progression of chronic liver disease (CLD) to HCC, thereby identifying the mechanisms relevant to HCC development. This hypothesis was tested in HBx transgenic (HBxTg) mice fed SCFAs. Groups of HBxTg mice were fed with SCFAs or vehicle from 6 to 9 months of age and then assessed for dysplasia, and from 9 to 12 months of age and then assessed for HCC. Livers from 12 month old mice were then analyzed for changes in gene expression by mass spectrometry-based proteomics. SCFA-fed mice had significantly fewer dysplastic and HCC nodules compared to controls at 9 and 12 months, respectively. Pathway analysis of SCFA-fed mice showed down-regulation of signaling pathways altered by HBx in human CLD and HCC, including those involved in inflammation, phosphatidylinositol 3-kinase, epidermal growth factor, and Ras. SCFA treatment promoted increased expression of the tumor suppressor, disabled homolog 2 (DAB2). DAB2 depresses Ras pathway activity, which is constitutively activated by HBx. SCFAs also reduced cell viability in HBx-transfected cell lines in a dose-dependent manner while the viability of primary human hepatocytes was unaffected. These unique findings demonstrate that SCFAs delay the pathogenesis of CLD and development of HCC, and provide insight into some of the underlying mechanisms that are relevant to pathogenesis in that they are responsive to treatment.