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.

Recombinant human growth hormone (rhGH), the mainstay of hGH replacement therapy, is injected daily for years to enable children to achieve normal stature. Daily rhGH injections are required because its serum-T1/2 is <20 minutes. Human trials testing T1/2-extending rhGH proteins are promising, but even weekly rhGH administration poses potential short and long-term toxicity risks, including exacerbation of diabetes or hypertension and potential for oncogenesis. Frequent hGH injections produce non-physiologic IGF-I profiles, (peak serum IGF-I frequently supra-normal, trough IGF-I concentrations at baseline). Therefore, a safe, effective, single administration hGH approach that maintains hGH and IGF-I serum concentrations within their respective therapeutic ranges for approximately one year could offer significant advantages. Accordingly, DNARx tested HEDGESTM, it’s non-viral, non-integrating, DNA-and liposome-based systemic, in vivo gene therapy approach to produce long-term hGH serum levels within the therapeutic range (1 -10 ng/ml) in immunocompetent mice. Previously, one systemic HEDGES-human granulocyte colony stimulating factor (hG-CSF, serum T1/2<3 hours) cDNA administration produced durable therapeutic hG-CSF serum levels (Science advances, 2019, 5, 2019). Importantly, simply by modifying selected aspects of HEDGES’s DNA vector and liposomal components, the duration of serum hG-CSF protein production was controllable over a broad temporal range. Long-term assessment of serum hGH in immunocompetent mice was assessed by injecting either one HEDGES administration or one administration followed by one re-dose of DNA vectors expressing either A) wildtype hGH cDNA (HEDGES-1) or B) wildtype hGH fused to selected serum protein T1/2-extending DNA sequences (HEDGES-2), with these results: 1) One HEDGES-1 administration produced slowly-increasing, but sub-therapeutic hGH serum levels from day 7 to day 99 after injection. From day 99 on, serum hGH levels remained within the therapeutic range for > 230 days; 2) Concurrently, hGH-induced elevation of endogenous mouse serum IGF-I levels remained between 1 to 3 fold above baseline. 3) One HEDGES-1 re-injection was administered 35 days after initial injection, a time when hGH serum levels remained sub-therapeutic. By day 7, one re-injection further elevated cDNA-encoded hGH serum levels into the therapeutic range, maintaining them for >175 days; 4) One HEDGES-2 administration produced therapeutic hGH serum levels by day 7 that were maintained for >250 days. These studies demonstrate the feasibility of achieving very long-term therapeutic levels of hGH and IGF-I following 1-2 injections. DNARx is now focused on selectively modifying its HEDGES hGH cDNA vectors and liposomal components to produce hGH serum levels within the therapeutic range for the equivalent of one human year following administration

AbstractMelanoma 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.

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 sub-type 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.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

Recombinant adeno-associated virus (AAV) vectors are transforming therapies for rare human monogenic deficiency diseases. However, adaptive immune responses to AAV and its limited DNA insert capacity, restrict their therapeutic potential. HEDGES (high-level extended duration gene expression system), a nonviral DNA- and liposome-based gene delivery platform, overcomes these limitations in immunocompetent mice. Specifically, one systemic HEDGES injection durably produces therapeutic levels of transgene-encoded human proteins, including FDA-approved cytokines and monoclonal antibodies, without detectable integration into genomic DNA. HEDGES also controls protein production duration from <3 weeks to >1.5 years, does not induce anti-vector immune responses, is reexpressed for prolonged periods following reinjection, and produces only transient minimal toxicity. HEDGES can produce extended therapeutic levels of multiple transgene-encoded therapeutic human proteins from DNA inserts >1.5-fold larger than AAV-based therapeutics, thus creating combinatorial interventions to effectively treat common polygenic diseases driven by multigenic abnormalities.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is expressed on the vascular endothelium and has been implicated in the late progression of metastatic tumors. The activity of PECAM-1 appears to be mediated by modulation of the tumor microenvironment (TME) and promotion of tumor cell proliferation, rather than through the stimulation of tumor angiogenesis. The present study aimed to extend those initial findings by indicating that the presence of functional PECAM-1 on the endothelium promotes a proliferative tumor cell phenotype in vivo, as well as in tumor cell (B16-F10 melanoma and 4T1 breast cancer cell lines) co-culture assays with mouse endothelial cells (ECs) or a surrogate EC line (REN-MP). The pro-proliferative effects were mediated by soluble endothelial-derived factors that were dependent on PECAM-1 homophilic ligand interactions, but which were independent of PECAM-1-dependent signaling. Further analysis of the conditioned media obtained from tumor/EC and tumor/REN-MP co-cultures identified TIMP metallopeptidase inhibitor-1 (TIMP-1) as a PECAM-1-regulated factor, the targeting of which in the tumor cell/REN-MP system inhibited tumor cell proliferation. In addition, TIMP-1 expression was decreased in metastatic tumors from the lungs of PECAM-1-null mice, thus providing evidence of the in vivo significance of co-culture studies. Taken together, these studies indicated that endothelial PECAM-1, through PECAM-1-dependent homophilic binding interactions, may induce release of TIMP-1 from the endothelium into the TME, thus leading to increased tumor cell proliferation.

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.

Abstract Targeted therapy relies on the classification of tumors according to the major molecular drivers of the malignant phenotype, which can then help decide the therapeutic treatment. However, a substantial subset of solid tumors does not express these markers, exemplified by the triple-negative subtype of breast cancer. Shared molecular factors that promote the progression of these tumors, and that represent a target for their therapy, are missing. We previously described a role for PHIP (Pleckstrin Homology domain-Interacting Protein) in the progression of melanoma, and demonstrated PHIP activation in triple-negative melanomas. Analysis of the TCGA profiling efforts in melanoma, breast and lung cancer revealed PHIP expression to be enriched in triple-negative breast cancer and in the bronchiad subtype of triple-negative lung cancer. Here we show the broad-based role of PHIP in the progression of triple-negative subtypes of three solid tumors (breast and lung cancer, and melanoma) defined by different mutational drivers and targeted therapies. By using a shRNA-based targeting of PHIP in in vitro and in vivo models, we have suppressed the malignant phenotype of triple-negative MDA-MB-231 and MDA-MB-436 human breast carcinoma cells. In addition, PHIP knockdown resulted in significant anti tumor effects in H1703 and Calu3 human lung cancer cells lacking mutations in EGFR, KRAS, and ALK, and in two short-term triple wild type melanoma cultures lacking mutations in BRAF, NRAS, and NF1. Suppression of PHIP expression resulted in inhibition of both tumor cell proliferation by cell survival and colony formation assays and invasion into matrigel in each of the tumor models examined, and was accompanied by suppression of pAKT, CCND1, TLN1, and ITGβ1 expression when assessed by western-blot or quantitative immunofluorescence. The PHIP protein contains two bromodomains that can be therapeutically targeted by small molecules. However, the functional activity of the PHIP bromodomains has been poorly characterized. We show that PHIP co-localizes with and binds to the acetylated histone modification H4K91ac, and that both are coordinately regulated upon growth factor stimulation suggesting a new function for PHIP as a chromatin remodeler. To conclude, our results identify a novel role for PHIP in the progression of solid tumors lacking the major molecular drivers, and suggest PHIP as a druggable target for the therapy of these malignancies. Citation Format: David De Semir, Vladimir Bezrookove, Mehdi Nosrati, Altaf A. Dar, Nathan Salomonis, Richard W. Sagebiel, Pierre Desprez, Robert J. Debs, Dirk Schadendorf, Mohammed Kashani-Sabet. PHIP is a therapeutic target for triple negative solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3031. doi:10.1158/1538-7445.AM2017-3031

Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.

BACKGROUND:Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we explore the oncogenic effects of BPTF in melanoma.METHODS:The consequences of differential expression of BPTF were explored using shRNA-mediated knockdown in several melanoma cell lines. Immunoblotting was used to assess the expression of various proteins regulated by BPTF. The functional role of BPTF in melanoma progression was investigated using assays of colony formation, invasion, cell cycle, sensitivity to selective BRAF inhibitors, and in xenograft models of melanoma progression (n = 12 mice per group). The biomarker role of BPTF in melanoma progression was assessed using fluorescence in situ hybridization and immunohistochemical analyses. All statistical tests were two-sided.RESULTS:shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF copy number (mean ≥ 3) was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival P = .03, and disease-specific survival P = .008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell subclones, indicating the continued presence of drug-responsive subclones within tumors demonstrating overall resistance to anti-BRAF agents.CONCLUSIONS:These studies demonstrate multiple protumorigenic functions for BPTF and identify it as a novel target for anticancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF.

PECAM‐1 mediates homophilic and heterophilic ligand binding as well as intracellular signaling. Although endothelial PECAM‐1 has been implicated in metastatic tumor cell proliferation, the mechanism of this involvement is unknown. Mutants of PECAM‐1 in which PECAM‐1‐dependent homophilic binding, heterophilic binding or intracellular signaling had been disabled by targeted mutations were generated, and then expressed in REN cells (an EC surrogate). The resulting cells were then studied in tumor co‐culture assays using B16 melanoma, LOX human melanoma and murine 4T1 breast cancer. For all three tumors, tumor cell proliferation was simulated 2‐4 fold in the presence of cells expressing PECAM‐1, an activity that was lost if homophilic but not heterophilic or intracellular signaling was disabled. Consistent with the presence of a soluble factor, conditioned media (CM) from the co‐culture of REN cells expressing wild type PECAM‐1 stimulated tumor cell proliferation. An antibody array analysis of CM from murine EC/tumor cell co‐cultures identified TIMP1 (a simulator of tumor cell proliferation) as a possible factor whose expression was significantly inhibited by anti‐PECAM‐1 antibody. Consistent with this, we found that TIMP1 was significantly increased in the presence of cells expressing mouse PECAM‐1. This stimulation of TIMP1 expression was lost if homophilic binding was disrupted but was preserved if heterophilic binding or intracellular signaling were disabled. These data suggest that in vivo, endothelial PECAM‐1 regulates metastatic tumor cell proliferation through processes that involve PECAM‐1‐dependent homophilic ligand interactions and the release of endothelial‐derived TIMP1.

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.

Abstract Objectives: Patients who die from cancer succumb to advanced metastatic progression (AMP). While targeted therapies are available that address tumor cell derived regulators of metastasis, few therapeutics are being developed to address the molecular mechanisms of AMP. Our group has previously shown that vascular endothelial cell (VEC) PECAM-1 is a critical molecular master switch which regulates AMP by controlling the release of VEC-derived, paracrine growth factors that drive its progression. Anti-PECAM-1 monoclonal antibody (mAb) effectively and safely treats even pre-terminal cancers in AMP-bearing mice. The current experiments were performed to assess whether, to what extent and how widely targeting PECAM-1 can improve overall survival in mice bearing advanced metastases. Methods: Groups of wildtype (WT) C57Bl/6 mice, together with age and gender matched PECAM-1 knockout (KO) mice were intravenously injected with 25,000 murine B16-F10 melanoma cells, 30,000 murine EL4 lymphoma cells or 1,000,000 murine MC38 colon cancer cells. We then injected matched groups of PECAM-1 KO vs WT mice with 1,000,000 B16-F10 cells, to model maximally aggressive AMP. All groups were assessed for overall survival, using IACUC approved guidelines. Results: In all cases, overall survival (OS) was significantly prolonged in PECAM-1 KO vs WT controls (Kaplan-Meier ranged from p = 0.018 to p = 0.0002). Targeting microenvironmental PECAM-1 was as effective in increasing OS in mice receiving the very high tumor burden of 1,000,000 B16-F10 cells as in mice receiving 25,000 cells, further confirming the efficacy of targeting PECAM-1 against the most advanced models of metastasis that can be achieved preclinically. Furthermore, anti-PECAM-1 mAb-based therapy of B16-F10 metastase was as effective as the PECAM-1 KO genotype in increasing OS. Conclusion: To date, many approved mAb- and small molecule-inhibitor based targeted-therapies have shown limited abilities to either prolong OS as single agents, or to effectively treat multiple different solid tumor types. These results suggest that anti-PECAM-1 mAb therapy, which specifically targets VEC and paracrine factors they produce, has the potential to significantly increase OS in the setting of lethal advanced metastatic progression, independent of tumor type. Anti-PECAM-1 mAb therapy is as effective as a PECAM-1 knockout in improving OS. Phase 1 clinical trials are planned within 2 years. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2847. doi:1538-7445.AM2012-2847

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.

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.