Bromodomain inhibition overcomes treatment resistance in distinct molecular subtypes of melanoma.

Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation-Induced DNA Damage and Melanoma Susceptibility.

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2-M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma. SIGNIFICANCE: This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.

PHIP drives glioblastoma motility and invasion by regulating the focal adhesion complex.

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

PHIP as a therapeutic target for driver-negative subtypes of melanoma, breast, and lung cancer.

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.

Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis.

Invasion and metastasis of aggressive breast cancer cells are the final and fatal steps during cancer progression. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Therefore, effective, targeted, and non-toxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, down-regulated Id-1 gene expression in aggressive human breast cancer cells in culture. Using cell proliferation and invasion assays, cell flow cytometry to examine cell cycle and the formation of reactive oxygen species, and Western analysis, we determined pathways leading to the down-regulation of Id-1 expression by CBD and consequently to the inhibition of the proliferative and invasive phenotype of human breast cancer cells. Then, using the mouse 4T1 mammary tumor cell line and the ranksum test, two different syngeneic models of tumor metastasis to the lungs were chosen to determine whether treatment with CBD would reduce metastasis in vivo. We show that CBD inhibits human breast cancer cell proliferation and invasion through differential modulation of the extracellular signal-regulated kinase (ERK) and reactive oxygen species (ROS) pathways, and that both pathways lead to down-regulation of Id-1 expression. Moreover, we demonstrate that CBD up-regulates the pro-differentiation factor, Id-2. Using immune competent mice, we then show that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis. Our data demonstrate the efficacy of CBD in pre-clinical models of breast cancer. The results have the potential to lead to the development of novel non-toxic compounds for the treatment of breast cancer metastasis, and the information gained from these experiments broaden our knowledge of both Id-1 and cannabinoid biology as it pertains to cancer progression.

Ribozyme-mediated targeting of IkappaBgamma inhibits melanoma invasion and metastasis.

IkappaBgamma is one member of a family of proteins that can inhibit the nuclear localization of nuclear factor-kappaB. However, the other specific functions of IkappaBgamma are still poorly understood, and its effects on tumor metastasis have not yet been characterized. We examined the consequences of targeting IkappaBgamma in melanoma cells using a hammerhead ribozyme. We developed stable transformant B16-F10 melanoma cell lines that express a ribozyme that targets mouse IkappaBgamma (IkappaBgamma-144-Rz). Tail-vein injection of B16-F10 cells that stably express IkappaBgamma-144-Rz into mice resulted in a significant reduction of the metastatic potential of these cells. IkappaBgamma-144-Rz-expressing B16 cells were shown to have increased transcriptional activity of nuclear factor-kappaB. We then showed that IkappaBgamma-144-Rz-expressing cells demonstrated both reduced invasion and increased apoptosis, suggesting the existence of pathways through which IkappaBgamma promotes melanoma metastasis. Using gene expression profiling, we identified a differentially expressed gene set that is regulated by the stable suppression of IkappaBgamma that may participate in mediating its anti-metastatic effects; we also confirmed the altered expression levels of several of these genes by quantitative real time polymerase chain reaction. Plasmid-mediated expression of IkappaBgamma-144-Rz produced a significant inhibition of the metastatic progression of B16-F10 cells to the lung and resulted in significant anti-invasive and pro-apoptotic effects on murine Lewis lung carcinoma cells. Our results suggest a novel role for IkappaBgamma in promoting the metastatic progression of melanoma.

Genes and pathways downstream of telomerase in melanoma metastasis.

Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.

Membrane-permeant, DNA-binding agents alter intracellular trafficking and increase the transfection efficiency of complexed plasmid DNA.

Nuclear delivery of extracellular DNA by nonviral vectors is inhibited by a series of cell membrane and compartmental barriers. Certain cationic amphiphiles that partition through cellular membranes to bind genomic DNA can enhance nuclear delivery of plasmid DNA. Specifically, delivering plasmid DNA complexed to the DNA-binding dye Hoechst 33258 produces cellular transfection levels similar to those achieved by cationic liposome:DNA complexes (CLDC), with less toxicity. Incorporating Hoechst into CLDC or polyethyleneimine:DNA complexes significantly increased reporter gene expression, as well as the percentage of cells transfected. Hoechst:CLDC significantly improved transfection of nondividing cells and efficiently transfected cells in the presence of anionic molecules that block cellular uptake of and transfection by CLDC alone. Hoechst:CLDC also increased gene expression in mouse tissues following intravenous delivery. Delivery of fluorescently labeled plasmid DNA via Hoechst altered its intracellular trafficking by both minimizing lysosomal sequestration and accelerating delivery into the nucleus. Agents such as Hoechst constitute a novel class of nonviral carriers that can confer their membrane-permeant properties on complexed DNA, thus redirecting its intracellular trafficking. In addition, binding of Hoechst 33258 to specific chromosomal DNA target sequences and its ability to modulate transcription may further enhance the expression of delivered genes.

Id genes and proteins as promising targets in cancer therapy.

Since the identification of Id proteins more than a decade ago, much work has demonstrated their regulatory roles in development, cell fate and lineage determination, proliferation, differentiation, angiogenesis, invasion and migration. Recent studies reveal not only that Id protein expression is significantly correlated both with cancer progression and with overall prognosis, but also that it can be exploited as a therapeutic target. This review will focus on the recent advances in our understanding of the relationships between Id expression and cancer, as well as providing a rationale for developing therapeutic strategies using Ids as targets to treat metastatic cancers.

Antitumor activity of systemically delivered ribozymes targeting murine telomerase RNA.

PURPOSE: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo. EXPERIMENTAL DESIGN: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol. RESULTS: Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice. CONCLUSIONS: Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.

The effect of liposome size on the final lipid/DNA ratio of cationic lipoplexes.

Several studies have demonstrated that lipoplexes are two-phase systems over most mixing lipid/DNA charge ratios. Because these studies have focused on small unilamellar vesicles (SUV), they leave open the question as to whether a similar pattern is followed by other liposome types. The main purpose of this work is to examine the question further by characterizing the assembly of cationic lipoplexes prepared from 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM)/dioleoylphosphatidylethanolamine (DOPE) (1:1) liposomes of various types. Sedimentation in sucrose density gradients reveals that large unilamellar vesicles (LUV) and sedimented multilamellar vesicles (sMLV), as opposed to SUV, form lipoplexes that exist as a single phase over a relatively broad range of mixing (+/-) ratios. This is indicated by observing that most of the LUV and sMLV become involved in the assembly reaction up to mixing (+/-) ratios of 4 and 9, respectively, while only a small and constant fraction of SUV associates with DNA at all mixing (+/-) ratios tested. Consequently, while maximal (+/-) ratios of approximately 4.5 and 9 are found in LUV and sMLV lipoplexes, respectively, a final (+/-) ratio of only approximately 2 is determined in SUV lipoplexes. Isothermal titration calorimetry shows that this is the lowest possible charge ratio achieved when liposomes are titrated with DNA. Based on these observations and on the size differences of the liposomes used, a model of lipoplex formation is proposed.

NF-kappa B in the vascular progression of melanoma.

PURPOSE: To examine a model of melanoma progression based on vascular factors and the role of NF-kappa B in the vascular progression of melanoma. PATIENTS AND METHODS: A data set of 526 patients from the University of California San Francisco Melanoma Center with 2 years of follow-up or first relapse was studied. The impact of the presence or absence of various prognostic factors on overall survival of melanoma patients was assessed using Cox regression and Kaplan-Meier analysis. A matched-pair analysis of NF-kappa B expression was performed in cases with vascular involvement and increased tumor vascularity versus matched controls lacking these factors. RESULTS: Cox regression analysis of factors evaluated by the American Joint Committee on Cancer Melanoma Staging Committee reproduced the powerful impact of tumor thickness and ulceration in this data set. With the inclusion of vascular factors such as tumor vascularity and vascular involvement, ulceration was no longer significant in predicting overall survival. By multivariate analysis, vascular involvement and tumor vascularity were the strongest predictors of melanoma outcome. Tumor vascularity seems to be a precursor of both vascular involvement and ulceration. A matched-pair tissue array analysis demonstrated the significant correlation between overexpression of NF-kappa B-p65 and the development of vascular factors. CONCLUSION: Vascular factors play an important role in the progression of malignant melanoma. Ulceration may be a surrogate marker for the interactions between melanoma and the tumor vasculature. NF-kappa B seems to play an important role in the development of these factors.

Functional identification of distinct sets of antitumor activities mediated by the FKBP gene family.

Assigning biologic function to the many sequenced but still uncharacterized genes remains the greatest obstacle confronting the human genome project. Differential gene expression profiling routinely detects uncharacterized genes aberrantly expressed in conditions such as cancer but cannot determine which genes are functionally involved in such complex phenotypes. Integrating gene expression profiling with specific modulation of gene expression in relevant disease models can identify complex biologic functions controlled by currently uncharacterized genes. Here, we used systemic gene transfer in tumor-bearing mice to identify novel antiinvasive and antimetastatic functions for Fkbp8, and subsequently for Fkbp1a. Fkbp8 is a previously uncharacterized member of the FK-506-binding protein (FKBP) gene family down-regulated in aggressive tumors. Antitumor effects produced by Fkbp1a gene expression are mediated by cellular pathways entirely distinct from those responsible for antitumor effects produced by Fkbp1a binding to its bacterially derived ligand, rapamycin. We then used gene expression profiling to identify syndecan 1 (Sdc1) and matrix metalloproteinase 9 (MMP9) as genes directly regulated by Fkbp1a and Fkbp8. FKBP gene expression coordinately induces the expression of the antiinvasive Sdc1 gene and suppresses the proinvasive MMP9 gene. Conversely, short interfering RNA-mediated suppression of Fkbp1a increases tumor cell invasion and MMP9 levels, while down-regulating Sdc1. Thus, syndecan 1 and MMP9 appear to mediate the antiinvasive and antimetastatic effects produced by FKBP gene expression. These studies show that uncharacterized genes differentially expressed in metastatic cancers can play important functional roles in the metastatic phenotype. Furthermore, identifying gene regulatory networks that function to control tumor progression may permit more accurate modeling of the complex molecular mechanisms of this disease.

Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis.

Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive in vitro and less metastatic in vivo when Id-1 is down-regulated by stable transduction with antisense Id-1. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted Id-1 expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target Id-1 expression to reduce breast cancer metastasis in humans.

Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice.

To date, functional genomic studies have been confined to either cell-based assays or germline mutations, using transgenic or knockout animals. However, these approaches are often unable either to recapitulate complex biologic phenotypes, such as tumor metastasis, or to identify the specific genes and functional pathways that produce serious diseases in adult animals. Although the transcription factor NF-kappaB transactivates many metastasis-related genes in cells, the precise genes and functional-pathways through which NF-kappaB regulates metastasis in tumor-bearing hosts are poorly understood. Here, we show that the systemic delivery of plasmid-based ribozymes targeting NF-kappaB in adult, tumor-bearing mice suppressed NF-kappaB expression in metastatic melanoma cells, as well as in normal cell types, and significantly reduced metastatic spread. Plasmid-based ribozymes suppressed target-gene expression with sequence specificity not achievable by using synthetic oligonucleotide-based approaches. NF-kappaB seemed to regulate tumor metastasis through invasion-related, rather than angiogenesis-, cell-cycle- or apoptosis-related pathways in tumor-bearing mice. Furthermore, ribozymes targeting either of the NF-kappaB-regulated genes, integrin beta(3) or PECAM-1 (a ligand-receptor pair linked to cell adhesion), reduced tumor metastasis at a level comparable to NF-kappaB. These studies demonstrate the utility of gene targeting by means of systemic, plasmid-based ribozymes to dissect out the functional genomics of complex biologic phenotypes, including tumor metastasis.

Distinct sets of cellular genes control the expression of transfected, nuclear-localized genes.

Gene transfection, a process used to study gene function, is itself poorly understood. Transfection-enhancing agents, including phorbol myristic acid (PMA) and histone deacetylase (HDAC) inhibitors, have been shown to increase transfection efficiency either by improving gene delivery into cells or by acting directly on delivered DNA sequences to increase their expression. Our results indicate that PMA and HDAC inhibitors can also regulate transfection efficiency by modulating distinct classes of cellular genes, which otherwise limit or block the expression of transfected genes already present in the nucleus. Either HDAC inhibitors or PMA was required to express reporter plasmids already present in the nucleus of lymphocyte lines. HDAC inhibitors and PMA seemed to operate through "transfection-controlling" cellular genes or gene products, rather than acting directly on transfected expression plasmids. PMA appeared to increase transfection efficiency by activating PKC-inducible, immediate-early gene products. Conversely, HDAC inhibitors functioned through a non-PKC-dependent pathway that required new protein synthesis, potentially acting through the de-repression of chromosomal genes. Neither delivery across the cell membrane nor into the nucleus may be rate-limiting for expressing transfected genes in some cell lines. In such cells, the targeted modulation of specific cellular genes may be required to efficiently express transfected genes.

Strain-based genetic differences regulate the efficiency of systemic gene delivery as well as expression.

We have characterized the impact of strain-based genetic differences on the efficiency of the intravenous cationic liposome-DNA complex (CLDC)-based gene transfer and expression in mice. We also investigated what steps in the gene delivery and expression pathway appeared responsible for these strain-related differences and whether such differences could be compensated for either by agents that alter host pathways important in CLDC-mediated gene transfer and expression, or by changes in CLDC formulation. We found that different mouse strains can exhibit different expression levels and/or differences in the amount of plasmid DNA delivered to the organs where the DNA is expressed. Furthermore, drug pretreatment or reformulation of the CLDC could improve DNA delivery and/or gene expression in a strain-specific fashion. We conclude that genetic factors critically modify both the tissue deposition and the expression of genetic materials delivered by CLDC. Because manipulation of either the host or the CLDC could at least partially compensate for these strain-related differences, such strategies may be required to effectively use non-viral gene transfer approaches in genetically diverse populations.