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THIS TEXT NOT FINISHED, UNDER CONSTRUCTION.<\/p>\n

\u201cFor years, studies have suggested cannabinoids [found in cannabis] fight different forms of breast cancer.\u201d
\nUltimately, as the title of the article stated, CBD modulates the tumor microenvironment to inhibit cancer through multiple mechanisms. It is important to note that EGF is slightly different than HER-2, and while the latter is always absent in triple-negative breast cancer, the former is often present.
\nAnother February 2015 study in the Journal of Medicinal Chemistry found that a synthetic cannabinoid induced programmed cell death in TNBC cell lines via CB2 receptor activation. When the compound was tested on normal tissue, no toxic effects were observed. Since THC activates CB2 receptors, and CBD has some weak affinity for it, those phytocannabinoids could also theoretically kill TNBC.
\nStudies Prove Viability of Cannabinoid Treatments
\nFor years, studies have suggested cannabinoids fight different forms of breast cancer. THC has been shown to induce apoptosis in ErbB2-positive breast cancer cells and inhibit tumor growth in animals. CBD is known to work at the genetic level, blocking expression of the ID-1 gene to inhibit breast cancer metastasis.
\nstefanie larueGiven these studies, it is no surprise that many humans have had direct success against their cancers with cannabis extracts. Stefanie LaRue beat Stage IV breast cancer during its third recurrence using cannabis oil. While chemotherapy and other techniques had stopped the disease twice before, they were apparently not enough to completely eliminate the cancer.
\n\u201cCannabis oil killed all of the tumors in my body. My monthly lab and quarterly scan results are proof that the cannabis oil treatment worked,\u201d Stefanie said. Other similar stories can be found online, although none seem to directly relate to TNBC. However, since extracts have reportedly been effective against numerous cases of Stage IV terminal breast cancer, it is likely some of them were TNBC. In any case, more research is desperately needed to determine the effectiveness of cannabis extracts against this aggressive breast cancer.<\/p>\n

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Modulation of the tumor microenvironment and inhibition of EGF\/EGFR pathway: Novel anti-tumor mechanisms of Cannabidiol in breast cancer<\/p>\n

Mohamad Elbazemail, Mohd W. Nasseremail, Janani Raviemail, Nissar A. Waniemail, Dinesh K. Ahirwaremail, Helong Zhaoemail, Steve Oghumuemail, Abhay R. Satoskaremail, Konstantin Shiloemail, William E. Carson IIIemail, Ramesh K. Ganjucorrespondenceemail
\nMolecular Oncology: \u00a0Accepted: December 27, 2014; Published Online: January 19, 2015
\nHighlights
\n\u2022CBD inhibits TNBC proliferation, migration and invasion.
\n\u2022CBD inhibits EGF\/EGFR signaling pathway and its downstream targets.
\n\u2022CBD inhibits GM-CSF, CCL3 and MIP-2 secretion from cancer cells.
\n\u2022CBD inhibits breast cancer growth and metastasis in two mouse models.
\n\u2022CBD inhibit M2 macrophage recruitment to the tumor stroma.<\/p>\n

Abstract<\/p>\n

The anti-tumor role and mechanisms of Cannabidiol (CBD), a non-psychotropic cannabinoid compound, are not well studied especially in triple-negative breast cancer (TNBC). In the present study, we analyzed CBD’s anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time-that CBD significantly inhibits epidermal growth factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor-associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of GM-CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows -for the first time-that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF\/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.<\/p>\n

Keywords:
\nCannabidiol, Tumor microenvironment, EGFR, Triple negative breast cancer
\nAbbreviations:
\nCBD (Cannabidiol), EGFR (epidermal growth factor receptor), TNBC (triple negative breast cancer), MMP (matrix metallo-proteinase), TAMs (tumor associated macrophages)<\/p>\n

http:\/\/www.moloncol.org\/article\/S1574-7891(14)00295-6\/fulltext<\/p>\n

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exhibit B<\/p>\n

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http:\/\/pubs.acs.org\/doi\/abs\/10.1021\/acs.jmedchem.5b00078<\/p>\n

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Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition
\nMar\u00eda M Caffarel16, Clara Andradas1, Emilia Mira2, Eduardo P\u00e9rez-G\u00f3mez1, Camilla Cerutti16, Gema Moreno-Bueno3, Juana M Flores4, Isabel Garc\u00eda-Real4, Jos\u00e9 Palacios5, Santos Ma\u00f1es2, Manuel Guzm\u00e1n1 and Cristina S\u00e1nchez1*<\/p>\n

* Corresponding author: Cristina S\u00e1nchez cristina.sanchez@quim.ucm.es<\/p>\n

Author Affiliations
\n1 Dept. Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain<\/p>\n

2 Dept. Immunology and Oncology, Centro Nacional de Biotecnolog\u00eda, Madrid, Spain<\/p>\n

3 Dept. Biochemistry, School of Medicine, Universidad Aut\u00f3noma-Instituto de Investigaciones Biom\u00e9dicas “Alberto Sols”, IdiPaz, Madrid, Spain<\/p>\n

4 Dept. Animal Surgery and Medicine, School of Veterinary, Complutense University, Madrid, Spain<\/p>\n

5 Servicio de Anatom\u00eda Patol\u00f3gica, Hospital Virgen del Roc\u00edo, Seville, Spain<\/p>\n

6 Current Address: MMC, Dept. Pathology, University of Cambridge, United Kingdom; CC, Dept. Life Sciences, The Open University, Milton Keynes, UK<\/p>\n

For all author emails, please log on.<\/p>\n

Molecular Cancer 2010, 9:196 doi:10.1186\/1476-4598-9-196<\/p>\n

The electronic version of this article is the complete one and can be found online at: http:\/\/www.molecular-cancer.com\/content\/9\/1\/196
\nReceived: 26 January 2010
\nAccepted: 22 July 2010
\nPublished: 22 July 2010
\n\u00a9 2010 Caffarel et al; licensee BioMed Central Ltd.
\nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:\/\/creativecommons.org\/licenses\/by\/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.<\/p>\n

Abstract
\nBackground
\nErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors.<\/p>\n

Results
\nOur results show that both \u03949-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount\/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2.<\/p>\n

Conclusions
\nTaken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer.<\/p>\n

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http:\/\/www.molecular-cancer.com\/content\/9\/1\/196<\/p>\n

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EXHIBIT D<\/p>\n

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Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells
\nSean D. McAllister, Rigel T. Christian, Maxx P. Horowitz, Amaia Garcia and Pierre-Yves Desprez
\n+ Author Affiliations<\/p>\n

California Pacific Medical Center, Research Institute, San Francisco, California
\nRequests for reprints: Sean D. McAllister, California Pacific Medical Center, Research Institute, 475 Brannan Street, San Francisco, CA 94107. Phone: 415-600-5926; Fax: 415-600-1725. E-mail: mcallis@cpmcri.org<\/p>\n

Next Section
\nAbstract<\/p>\n

Invasion and metastasis of aggressive breast cancer cells is the final and fatal step during cancer progression, and is the least understood genetically. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Clearly, effective and nontoxic 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. Using a mouse model, we previously determined that metastatic breast cancer cells became significantly less invasive in vitro and less metastatic in vivo when Id-1 was down-regulated by stable transduction with antisense Id-1. It is not possible at this point, however, to use antisense technology to reduce Id-1 expression in patients with metastatic breast cancer. Here, we report that cannabidiol (CBD), a cannabinoid with a low-toxicity profile, could down-regulate Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive phenotype of breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. These effects seemed to occur as the result of an inhibition of the Id-1 gene at the promoter level. Importantly, CBD did not inhibit invasiveness in cells that ectopically expressed Id-1. In conclusion, CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the down-regulation of tumor aggressiveness. [Mol Cancer Ther 2007;6(11):2921\u20137]\n

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