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 pro-file, 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.

Glioblastoma (GBM) is the most common form of primary adult brain tumors. A majority of GBMs grow invasively into distant brain tissue, leading to tumor recurrence, which is ultimately incurable. It is, therefore, essential to discover master regulators that control GBM invasiveness and target them therapeutically. We demonstrate here that the transcriptional regulator Id-1 plays a critical role in modulating the invasiveness of GBM cell lines and primary GBM cells. Id-1 expression levels positively correlate with glioma cell invasiveness in culture and with histopathological grades in patient biopsies. Id-1 knockdown dramatically reduces GBM cell invasion that is accompanied by profound morphological changes and robust reduction in expression levels of “mesenchymal” markers, as well as inhibition of self-renewal potential and down-regulation of glioma stem cell markers. Importantly, genetic knockdown of Id-1 leads to a significant increase in survival in an orthotopic model of human GBM. Furthermore, we show that a non-toxic compound, cannabidiol, significantly down-regulates Id-1 gene expression and associated glioma cell invasiveness and self-renewal. Additionally, cannabidiol significantly inhibits the invasion of GBM cells through an organotypic brain slice and glioma progression in vivo. Our results suggest that Id-1 regulates multiple tumor-promoting pathways in GBM, and that drugs targeting Id-1 represent a novel and promising strategy for improving the therapy and outcome of GBM patients.

BACKGROUND AND PURPOSEThe psychoactive cannabinoid Δ 9 -tetrahydrocannabinol (THC) and the non-psychoactive cannabinoid cannabidiol (CBD) can both reduce cancer progression, each through distinct anti-tumour pathways. Our goal was to discover a compound that could efficiently target both cannabinoid anti-tumour pathways. EXPERIMENTAL APPROACHTo measure breast cancer cell proliferation/viability and invasion, MTT and Boyden chamber assays were used. Modulation of reactive oxygen species (ROS) and apoptosis was measured using dichlorodihydrofluorescein and annexin/propidium iodide, respectively, in combination with cell flow cytometry. Changes in protein levels were evaluated using Western analysis. Orthotopic and i.v. mouse models of breast cancer metastasis were used to test the activity of cannabinoids in vivo. KEY RESULTSCBD reduced breast cancer metastasis in advanced stages of the disease as the direct result of down-regulating the transcriptional regulator Id1. However, this was associated with moderate increases in survival. We therefore screened for analogues that could co-target cannabinoid anti-tumour pathways (CBD-and THC-associated) and discovered the compound O-1663. This analogue inhibited Id1, produced a marked stimulation of ROS, up-regulated autophagy and induced apoptosis. Of all the compounds tested, it was the most potent at inhibiting breast cancer cell proliferation and invasion in culture and metastasis in vivo. CONCLUSIONS AND IMPLICATIONSO-1663 prolonged survival in advanced stages of breast cancer metastasis. Developing compounds that can simultaneously target multiple cannabinoid anti-tumour pathways efficiently may provide a novel approach for the treatment of patients with metastatic breast cancer.

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The purpose of this study is to establish a tumor marker that can be applied for the early detection and follow-up of oral cancer patients. Employing the proteomic approach using MALDI TOF-MS, 2-DE, patient's sera and culturing cell lines, the serum autoantibodies (autoAbs) were screened and the serum levels were estimated by ELISA. Targeting the tumor cell invasion into the surrounding stromal tissues, MRC-5 human fibroblasts were employed as the target cells and a mitochondrial membrane protein, sideroflexin 3 (SFXN3), was identified. The serum anti-SFXN3-autoAb levels elevated in patients with the oral squamous cell carcinoma significantly: with 77% sensitivity and 89% specificity against control samples. The serum anti-SFXN3-autoAb levels were mildly correlated with the primary tumor sizes, however, the levels were slightly highly elevated in T1 early cancer. An immunohistochemical analysis revealed that the SFXN3 protein is expressed in the stromal fibroblasts between the caner nests and also in the basal layer of the squamous epithelium. Changes in the serum anti-SFXN3-autoAb levels after therapy correlated with the clinical tumor burden. These findings demonstrated that the serum anti-SFXN3-autoAb is worthy of clinical evaluation as a potentially of the novel tumor maker for the early detection of oral squamous cell carcinoma.

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