Although the concept of cancer stem cells (CSCs) is well-accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5′ to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines. [Cancer Res 2008;68(5):1362–8]

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (ER⁺) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer–associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Experimental Design: Tissues from patients with ER⁺ breast cancer were analyzed for the presence of CD146-positive (CD146^pos) and CD146-negative (CD146^neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER⁺ tumor cells cocultured with CD146^pos or CD146^neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER⁺ breast cancer. Results: We demonstrate that ER⁺ breast cancers contain two CAF subtypes defined by CD146 expression. CD146^neg CAFs suppress ER expression in ER⁺ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146^pos CAFs maintains ER expression in ER⁺ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146^pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146^neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER⁺ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

Immune checkpoint inhibitors have improved overall survival rates for many cancers, yet the majority of patients do not respond to treatment and succumb to disease progression. One tumor-related mechanism limiting the efficacy of immunotherapies in melanoma is the recruitment and expansion of myeloid-derived suppressor cells (MDSCs). Therefore, targeting MDSCs in combination with immunotherapies is an attractive strategy to improve response rates and effectiveness. We tested this strategy by designing a randomized phase II clinical trial treating advanced melanoma patients with either Ipilimumab monotherapy or Ipilimumab plus all-trans retinoic acid (ATRA). Clinicaltrails.gov identifier (NCT02403778). The frequency of circulating MDSCs and the activation of CD8(+) T cells was measured by flow cytometry. Expression of immunosuppressive genes was measured with quantitative real time-PCR. T cell suppressive functions were measured by mixed lymphocyte reaction. Here we show that in vitro treatment with ATRA decreases immunosuppressive function of MDSCs in mixed lymphocyte reactions. Additionally, ATRA reduces the expression of immunosuppressive genes including PD-L1, IL-10, and indoleamine 2,3‑dioxygenase by MDSCs. Furthermore, the addition of ATRA to standard of care Ipilimumab therapy appears safe, as ATRA did not increase the frequency of grade 3 or 4 adverse events. Finally, ATRA significantly decreased the frequency of circulating MDSCs compared to Ipilimumab treatment alone in advanced-stage melanoma patients. These results illustrate the importance of MDSCs in immunotherapy resistance and provide evidence that targeting MDSCs in cancer patients may augment immunotherapeutic approaches.