Indole-3-carbinol (I3C), a key bioactive product of Glucobrassicin, found in cruciferous vegetables has been demonstrated to control the formation, growth and metastasis of certain human cancers with minimal side effects especially during prolonged treatments. It is currently in clinical trials for the prevention and treatment of reproductive cancers such as breast and prostrate cancer. However, relatively little is known about the effects of I3C on human skin cancer.
Human melanoma, the most aggressive form of skin cancer currently poses a big clinical challenge due to the inevitable development of resistance to currently available therapies, and has a dismal 5year survival rate. Approximately 50-60% of melanomas contain an oncogenic mutation in the B-RAF gene that leads to constitutive activation of its downstream proliferative signaling via the MAP kinase pathway. One therapeutic strategy that showed significant promise in preclinical and clinical trials was the selective targeting of the mutant BRAF in melanoma by synthetic B-RAF inhibitors like Vemurafenib or Dabrafenib. Unfortunately the long-term prognosis of metastatic melanoma patients treated with these oncogenic BRAF inhibitors remains poor due to the emergence of early or acquired resistance to these drugs and an increased risk for cancerous or precancerous non-melanoma skin lesions.
Efforts to unravel the molecular mechanisms leading to development of resistance to BRAF inhibitors have uncovered a complex network of signal transduction pathways that get activated and cross talk with each other to circumvent the inhibition of the BRAF signaling pathway. Some of the reported mechanisms of acquisition of BRAF inhibitor resistance include the homozygous loss of the tumor suppressor protein PTEN, a compensatory amplification of the lineage specific master regulator of melanoma biology Micropthalmia Associated Transcription Factor–Isoform M (MITF-M), as well as elevated levels of the Wnt proliferative signaling pathway. Therefore there is an emerging need in the field of melanoma-targeted therapy for drugs either with multiple mechanisms of actions which, can inhibit more than one of these oncogenic pathways or molecules that can be effectively combined with clinically used drugs to bring about a greater antiproliferative response with minimal levels of toxicity.
In this context, I3C was found to induce significant G1-phase cell-cycle arrest and apoptosis by stabilization of PTEN in human melanoma cells that express wild-type PTEN. This effect was not observed in cells with mutant or null PTEN genotypes neither did I3C have any detrimental affect on normal human epidermal melanocytes. In wild-type PTEN-expressing melanoma cell xenografted tumors formed in athymic nude mice, I3C attenuated growth of the tumors in vivo and the residual tumors showed increased levels of PTEN protein. Mechanistically, I3C disrupted the ubiquitination of PTEN by NEDD4-1, which prevented the proteasome-mediated degradation of PTEN without altering its transcript levels. RNAi-mediated knockdown of PTEN prevented the I3C-induced apoptotic response, whereas knockdown of NEDD4-1 mimicked the I3C apoptotic response, stabilized PTEN protein levels, and downregulated phosphorylated AKT-1 levels. Co-knockdown of PTEN and NEDD4-1 revealed that I3C- regulated apoptotic signaling through NEDD4-1 requires the presence of the wild-type PTEN protein. Finally, in silico structural modeling, in combination with isothermal titration calorimetry analysis, demonstrated that I3C directly interacts with purified NEDD4-1 protein. This study for the first time identified NEDD4-1 as a direct binding target of I3C in human melanoma.
However the antiproliferative action of I3C was found to be not restricted to melanoma cells lines expressing wild-type PTEN only. Oncogenic BRAF-V600E expressing melanoma cells were sensitive to I3C too, both in cell culture and in-vivo, independent of the status of PTEN. However in cell lines with a mutant BRAF and wild type PTEN the antiproliferative effect of I3C was found to be the maximum. In contrast, wild type BRAF-expressing melanoma cells remained relatively insensitive to I3C anti-proliferative signaling. In BRAF-V600E-expressing cells as well as in vivo, I3C strongly inhibited phosphorylation of the downstream effectors of BRAF signaling namely MEK, MAPK, and downregulated protein levels of BRN2, the transcription factor that mediates control of the BRAF signaling pathway over the melanocyte master regulator - MITF-M. Consequently upon treatment with I3C, MITF-M protein and transcripts levels were strongly attenuated with a significant inhibition of promoter activity and loss of endogenous BRN-2 binding to the MITF-M promoter. In silico modeling using known crystallographic structures of BRAF proteins predicted a thermodynamically stable I3C binding site within the HRD motif in the BRAF-V600E catalytic loop, whereas only low affinity interactions outside the catalytic domain were predicted with the wild type BRAF. In vitro kinase assays using immunoprecipitated BRAF-V600E and wild type BRAF demonstrated that I3C directly and selectively inhibited the enzymatic activity of only the oncogenic BRAF but not of the wild type protein.
Compared to the effects of each compound alone, combinations of I3C and the clinically used BRAF-V600E inhibitor Vemurafenib cooperatively inhibited melanoma cell proliferation and reduced MITF-M levels in BRAF-V600E expressing melanoma cells. Taken together, these results identified oncogenic BRAF-V600E as a key cellular target of I3C in human melanoma. Overall these studies revealed I3C as a rare small molecule with mechanisms of action targeting two key oncogenic signaling pathways in melanoma and thereby a potential candidate for novel single or combinatorial therapies to treat human melanoma in the clinic.
1-benzyl I3C, a synthetic analog previously demonstrated to be approximately 1000-fold more potent in its antiproliferative effect in human breast cancer cells in –vitro, was tested in an endeavor to find better, more potent anti-cancer compounds against melanoma. 1-benzyl I3C induced an antiproliferative effect and a G1 cell cycle arrest in melanoma cells in culture and attenuated xenografted tumor growth in-vivo in a murine model of melanoma, at 10 fold lower doses, at concentrations similar to currently used melanoma drugs. Additionally, 1-benzyl- I3C was effective against melanoma cells with a wider range of mutation profiles than I3C. Similar to I3C, 1-benyzl I3C significantly downregulated protein and transcript levels of MITF-M, but through the Wnt>GSK3β>β-catenin pathway which, is reportedly activated in one-third human melanoma specimens. 1-benzyl I3C downregulated protein levels of the Wnt co-receptor LRP6 leading to downstream release of inhibition on the β-catenin inhibitory complex, and consequent upregulation of one of the inhibitory proteins GSK3β, causing decrease in both cytoplasmic and nuclear β-catenin. Less nuclear β-catenin resulted in attenuation of MITF-M promoter activity accounting for the observed downregulation of MITF-M transcripts upon treatment with 1-benzyl I3C. Top Flash assays confirmed the involvement of the Wnt>GSK3β>β-catenin pathway in 1-benzyl Indole-3-carbinol’s mechanism of action. Combinations of 1-benzyl I3C with Vemurafenib used to treat melanoma cells, displayed a cooperative antiproliferative effect with a concomitant downregulation of MITF-M transcripts greater than each compound alone. This study identified 1-benzyl I3C as a novel synthetic inhibitor of Wnt signaling in melanoma that could be a promising mono or combinatorial therapeutic agent for human melanoma.