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618 Topical application of cyclic AMP modulators regulates melanoma tumor development and tumor growth
ABSTRACTS | Pigmentation & Melanoma 618
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Topical application of cyclic AMP modulators regulates melanoma tumor development and tumor growth CI Rodriguez1,2 and V Setaluri1,3,2 1 Dermatology, University of Wisconsin - Madison, Madison, WI, 2 Molecular and Environmental Toxicology, University of Wisconsin Madison, Madison, WI and 3 William S. Middleton Memorial Veterans Hospital, Madison, WI Cyclic AMP (cAMP) signaling, which is activated by binding of melanocyte stimulating hormone to melanocortin-1 receptor (MC1R), is essential for melanocyte proliferation and differentiation. MC1R gene polymorphisms cause cAMP signaling impairment and are associated with the red hair color and fair skin phenotype and higher risk of melanoma. However, the exact role of cAMP signaling in melanoma is not well understood. Here, we investigated the role of cAMP signaling in melanoma tumor development and growth using an inducible Braf (V600E) melanoma mouse model. To modulate cAMP levels in melanocytes, we applied topically DMSO (control) or the adenylyl cyclase (AC) activator forskolin (FSK) or inhibitor SQ 22536 (SQ) for two weeks prior or after the induction of tumor development by 4-hydroxytamoxifen (4HT). Tumor development was monitored for up to 45 days and tumors were excised for histological and molecular analysis. Our data show that decreasing cAMP inhibited the growth kinetics whereas elevating cAMP significantly accelerated the tumor growth and size. Histological analysis of the lesions showed that in the control mice, there was a marked peri-follicular proliferation of highly pigmented melanocytes and non-pigmented cells in the dermis. Similarly, in SQ treated mice, there was more extensive peri- and interfollicular accumulation of darkly pigmented cells with less extensive proliferation of non-pigmented cells deeper in the dermis. In contrast, the skin lesions from FSK treated mice showed proliferation of cells with less visible pigmentation, but cellular atypia, closer to the epidermis with little or no evidence of peri-follicular accumulation of melanocytes. Interestingly, we noted that inhibition of AC, but not its stimulation, results in accumulation of darkly pigmented melanocytes. In conclusion, our data suggest that cAMP promotes tumor development and growth after malignant transformation of melanocytes.
Altered expression of metabolic markers in cutaneous melanoma cells M Alonso1, L Najera2, E Carrasco3, N Salazar3, S Gonzalez4, Y Gilaberte5, J Cuezva6, F Rojo1 and A Juarranz3 1 Anatomı´a Patolo´gica, IIS-Fundacio´n Jime´nez Dı´az, Madrid, Spain, 2 Anatomı´a Patolo´gica, Hospital Universitario Puerta de Hierro, Madrid, Spain, 3 Biologı´a, Universidad Auto´noma of Madrid, Madrid, Spain, 4 Sloan Kettering Cancer Center, New York, NY, 5 Hospital San Jorge, Huesca, Spain and 6 Centro de Biologı´a Molecular “Severo Ochoa”, Madrid, Spain While normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, cancer cells change this energetic metabolism to an aerobic glycolysis (Warburg effect). Here, we have evaluated the expression of the b-subunit of the H+-ATP synthase (b-F1-ATPase), heat-shock protein 60 (Hsp60), glyceraldehyde3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) as metabolic markers in a panel of melanoma cell lines with different malignant phenotypes: C8161-HA (High Aggressive), C8161-PA (Poor Aggressive), C81-61 WM278 (vertical growth phase, VGP) and WM35 (radial growth phase, RGP). The expression of the metabolic markers was evaluated at both, mRNA and protein level by using RT-PCT and western blot, respectively. The bioenergetic cellular index (BEC), which represents a helpful biomarker of glucose utilization by tumor cells, was calculated from the expression of b-F1-ATPase, relative to the expression of the Hsp60 and the GAPDH. The results obtained indicate a decrease in the BEC, at both mRNA as well as protein level, with progressive aggressiveness of the studied cells. We have also observed a decrease in the BECs when melanoma cells are cultured as melanospheres compared with bidimensional cultures. As conclusion, energetic metabolism is in vitro dysregulated in melanoma, and mitochondrial and glycolytic alterations are associated with the stage of the disease, suggesting their importance in malignant biology and their potential utility as prognostic markers and biological target in melanoma.
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Soluble DC-HIL receptor from melanoma promotes metastasis by creating local immunosuppression at perivascular regions in lung V Ramani, T Teshima, J Chung and K Ariizumi Dermatology, The University of Texas Southwestern Medical Center, Dallas, TX Metastasis is the most dangerous aspect of melanoma. Primary tumors prepare microenvironment (pre-metastatic niches) in the distal organ for settlement of migratory tumor cells before metastasis by secreting soluble factors. However, the niche-formation and the role of T cells in this event remain ambiguous. B16 melanoma expresses T cell-inhibitory receptor DC-HIL and releases the soluble form (sDC-HIL) into the blood circulation, finally accumulating in lung before metastasis. To examine the direct effect on metastasis, mice were i.p. infected with sDC-HIL-V5- or GFP-lentiviruses. Blood sDC-HIL expression lasted for >4 wks. The infected mice were i.v. injected with LL2-neo tumor cells (no DC-HIL expressed), and lung metastasis was counted by clonogenic assay. Colonies of LL2-neo in mice expressing sDC-HIL was 6-fold more than in mice expressing GFP. This effect was reversed (70%) by injection of anti-V5 Ab a day before LL2 cell injection. In spontaneous metastasis, sDC-HIL-V5-transfected LL2 s.c. tumors produced 7-fold more colonies-lung than GFP-LL2 tumors. Relative location of sDC-HIL-accumulated spots (by anti-V5) to LL2 micrometastases (with CSFE) in lung was studied. Surprisingly, sDC-HIL accumulated at only few CD31+ endothelial cells EC (perivascular), in which LL2 tumors were colocalized. Binding of sDC-HIL to the SVEC line led us to examine if EC-bound sDC-HIL is T cell-suppressive. sDC-HIL-coated SVEC markedly suppressed CD8+ T cell activation (by 80%), which was completely reversed by anti-DC-HIL Ab. We then examined this inhibitory effect in vivo. Mice with sDC-HIL-V5 or GFP tumor received i.v. injection of CFSE-labeled activated CD8+ T cells, and 3 d later, the labeled T cells were assayed by FACS. CD8+ T cells in lung with sDC-HIL expression had significantly less # of proliferating cells than control (12% in sDC-HIL vs. 80% in GFP). Thus, the local accumulation of sDC-HIL onto particular EC makes the microenvironment immunosuppressive, a place that may serve as a pre-metastatic niche.
Down-regulated mir-23a contributes to invasion and metastasis of cutaneous melanoma by promoting autophagy Q Shi, H Wang, W Guo, Y Yang, W Zhang, S Guo, T Zhao, L Liu, Z Jian, Q Luan, L Liu, G Wang, T Gao and C Li Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Since the progression of melanoma from primary stage to advanced stage leads to a precipitous drop in the patients’ survival, the mechanisms underlying this process are of great importance. Recently, autophagy has been implicated in modulating cancer invasion and metastasis, and the level of autophagy has been found dysregulated in melanoma. Here, we reported miR-23a as a novel autophagy-regulating microRNA that played a remarkable role in modulating melanoma invasive and metastatic capacity. We found that serum miR-23a level was significantly down-regulated in melanoma patients and highly correlated with poor clinical outcomes. Notably, tissue miR-23a level was drastically decreased in metastatic melanoma compared with that in primary melanoma and melanocytic nevus. Furthermore, ectopic miR-23a expression prevented the invasion-metastasis cascade of melanoma both in vitro and in vivo, and overexpressed miR-23a suppressed the invasive and migratory property of melanoma by abrogating autophagy through directly targeting ATG12. Specially, attenuated melanoma invasion and migration mediated by overexpressed miR-23a resulted from the inhibition on ROS-HIF1a-RhoE signaling. Finally, we revealed that the down-regulation of miR-23a in metastatic melanoma was caused by RUNX2 in a transcriptional repressionmanner. Taken together, our findings demonstrate that miR-23a can act as a crucial epigenetic repressor of melanoma invasion and metastasis in an autophagy-dependent way, which indicates that miR-23a-mediated autophagy inhibition can be exploited to restrain invasionmetastasis cascade in melanoma treatment.
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CD8 to nearest tumor cell distance predicts disease progression in primary intermediate thickness melanoma B Dyring-Andersen1, J Lin1, M Dyring-Andersen1, JT O’Malley1, J Teague1, A Gehad1, C Hoyt2, PA Vieyra1 and R Clark1 1 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA and 2 PerkinElmer, Hopkinton, MA One of the clinical challenges in melanoma is the early identification of patients with intermediate risk melanomas who will go on to develop progressive disease. Effective combination immune checkpoint therapies are now available that can induce regression of even advanced melanoma. An important next step would be to use these therapies early in patients with primary melanomas at high risk for recurrence. A cohort of 30 primary intermediate risk cutaneous malignant melanomas (Breslow thickness 1-2 mm) were divided in two groups depending on sentinel lymph node biopsy (SLNB) status and subsequently thickness matched, resulting in 9 pairs. For pilot studies, six thickness matched tumors were included, 3 tumors from patients with positive SLNB and 3 tumors from patients with negative SLNB. We studied these primary tumors by multiplexed tyramide signal amplification based staining for CD4, CD8, FoxP3 and S100 expression and DAPI, followed by image deconvolution and automated cell analysis. Patients with a negative SLNB had a greater number of CD8+ T cells in tumors than patients with positive SLNB (202 vs. 98, p¼0.04). The numbers of FoxP3+ regulatory T cells and total CD4+ T cells were not significantly different. In breast cancer, the distance between CD8+ T cells and the nearest tumor cells identified patients who would develop progressive disease. We carried out nearest neighbor analysis on melanoma and found that the CD8+ T cell to nearest tumor cell distance was higher in tumors from patients with positive SLNB compared to patients with negative SLNB (120 vs. 70, p¼0.02). We are in the process of confirming this finding in the primary tumors in our initial cohort, as well as in 43 additional primary tumors with data on 5 year survival. In summary, we find that CD8+ T cell density and, more significantly, CD8+ T cell to tumor cell distance predicts progressive disease in primary intermediate risk melanoma.
S110 Journal of Investigative Dermatology (2016), Volume 136
CRD-BP as a potential prognostic marker and therapeutic target in melanoma YG Xu1, T Kim1,2, V Setaluri1 and V Spiegelman1,3 1 Dermatology, University of Wisconsin, Madison, WI, 2 Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI and 3 Department of Pediatrics, Pennsylvania State University, Hershey, PA CRD-BP is an RNA binding protein that binds to various targets such as mRNAs of bTrCP1, c-myc, Gli1, MDR1 and MITF, and affects their translation, localization and stability leading to increased survival, inhibition of apoptosis and development of drug resistance as well as induction of tumorigenic properties in a number of malignancies including melanoma. Current clinical staging of melanoma has missed a subset of thin melanomas that would progress aggressively and even cause death. Existing targeted molecular therapy such as inhibitors of V600E-mutant BRAF and combined inhibitors of BRAF and MARK is hampered by a short median length of response of only about 6 months, resistance to treatments, and no significant impact on overall survival. To explore the prognostic and therapeutic potential of CRD-BP, we have assessed the correlation between elevated CRD-BP expression and melanoma aggressiveness and clinical outcome using the melanoma tumor microarray and the prognostic and diagnostic significance of CRD BP over-expression in melanoma is discussed. Moreover, we have previously reported that inhibition of CRD-BP suppress melanoma growth regardless of BRAF mutation status. We now further demonstrate that in BRAFV600E melanoma cells sensitive to BRAF inhibitor, dual inhibition of mutant BRAF and CRD-BP is significantly more effective than single treatment (p<0.01). In cells with acquired or intrinsic resistance, dual inhibition no longer has additional benefit but down-regulation of CRD-BP alone still significantly inhibits cell growth (p<0.01). Similar results are seen in shortterm melanoma cultures freshly grown from patients, which are biologically more close to human melanoma than established cell lines. In BRAF-mutant melanomas, CRD-BP may be an effective adjuvant therapy to BRAF inhibitors and a novel strategy when resistance develops.
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Topical application of cyclic AMP modulators regulates melanoma tumor development and tumor growth CI Rodriguez1,2 and V Setaluri1,3,2 1 Dermatology, University of Wisconsin - Madison, Madison, WI, 2 Molecular and Environmental Toxicology, University of Wisconsin Madison, Madison, WI and 3 William S. Middleton Memorial Veterans Hospital, Madison, WI Cyclic AMP (cAMP) signaling, which is activated by binding of melanocyte stimulating hormone to melanocortin-1 receptor (MC1R), is essential for melanocyte proliferation and differentiation. MC1R gene polymorphisms cause cAMP signaling impairment and are associated with the red hair color and fair skin phenotype and higher risk of melanoma. However, the exact role of cAMP signaling in melanoma is not well understood. Here, we investigated the role of cAMP signaling in melanoma tumor development and growth using an inducible Braf (V600E) melanoma mouse model. To modulate cAMP levels in melanocytes, we applied topically DMSO (control) or the adenylyl cyclase (AC) activator forskolin (FSK) or inhibitor SQ 22536 (SQ) for two weeks prior or after the induction of tumor development by 4-hydroxytamoxifen (4HT). Tumor development was monitored for up to 45 days and tumors were excised for histological and molecular analysis. Our data show that decreasing cAMP inhibited the growth kinetics whereas elevating cAMP significantly accelerated the tumor growth and size. Histological analysis of the lesions showed that in the control mice, there was a marked peri-follicular proliferation of highly pigmented melanocytes and non-pigmented cells in the dermis. Similarly, in SQ treated mice, there was more extensive peri- and interfollicular accumulation of darkly pigmented cells with less extensive proliferation of non-pigmented cells deeper in the dermis. In contrast, the skin lesions from FSK treated mice showed proliferation of cells with less visible pigmentation, but cellular atypia, closer to the epidermis with little or no evidence of peri-follicular accumulation of melanocytes. Interestingly, we noted that inhibition of AC, but not its stimulation, results in accumulation of darkly pigmented melanocytes. In conclusion, our data suggest that cAMP promotes tumor development and growth after malignant transformation of melanocytes.
Altered expression of metabolic markers in cutaneous melanoma cells M Alonso1, L Najera2, E Carrasco3, N Salazar3, S Gonzalez4, Y Gilaberte5, J Cuezva6, F Rojo1 and A Juarranz3 1 Anatomı´a Patolo´gica, IIS-Fundacio´n Jime´nez Dı´az, Madrid, Spain, 2 Anatomı´a Patolo´gica, Hospital Universitario Puerta de Hierro, Madrid, Spain, 3 Biologı´a, Universidad Auto´noma of Madrid, Madrid, Spain, 4 Sloan Kettering Cancer Center, New York, NY, 5 Hospital San Jorge, Huesca, Spain and 6 Centro de Biologı´a Molecular “Severo Ochoa”, Madrid, Spain While normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, cancer cells change this energetic metabolism to an aerobic glycolysis (Warburg effect). Here, we have evaluated the expression of the b-subunit of the H+-ATP synthase (b-F1-ATPase), heat-shock protein 60 (Hsp60), glyceraldehyde3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) as metabolic markers in a panel of melanoma cell lines with different malignant phenotypes: C8161-HA (High Aggressive), C8161-PA (Poor Aggressive), C81-61 WM278 (vertical growth phase, VGP) and WM35 (radial growth phase, RGP). The expression of the metabolic markers was evaluated at both, mRNA and protein level by using RT-PCT and western blot, respectively. The bioenergetic cellular index (BEC), which represents a helpful biomarker of glucose utilization by tumor cells, was calculated from the expression of b-F1-ATPase, relative to the expression of the Hsp60 and the GAPDH. The results obtained indicate a decrease in the BEC, at both mRNA as well as protein level, with progressive aggressiveness of the studied cells. We have also observed a decrease in the BECs when melanoma cells are cultured as melanospheres compared with bidimensional cultures. As conclusion, energetic metabolism is in vitro dysregulated in melanoma, and mitochondrial and glycolytic alterations are associated with the stage of the disease, suggesting their importance in malignant biology and their potential utility as prognostic markers and biological target in melanoma.
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Soluble DC-HIL receptor from melanoma promotes metastasis by creating local immunosuppression at perivascular regions in lung V Ramani, T Teshima, J Chung and K Ariizumi Dermatology, The University of Texas Southwestern Medical Center, Dallas, TX Metastasis is the most dangerous aspect of melanoma. Primary tumors prepare microenvironment (pre-metastatic niches) in the distal organ for settlement of migratory tumor cells before metastasis by secreting soluble factors. However, the niche-formation and the role of T cells in this event remain ambiguous. B16 melanoma expresses T cell-inhibitory receptor DC-HIL and releases the soluble form (sDC-HIL) into the blood circulation, finally accumulating in lung before metastasis. To examine the direct effect on metastasis, mice were i.p. infected with sDC-HIL-V5- or GFP-lentiviruses. Blood sDC-HIL expression lasted for >4 wks. The infected mice were i.v. injected with LL2-neo tumor cells (no DC-HIL expressed), and lung metastasis was counted by clonogenic assay. Colonies of LL2-neo in mice expressing sDC-HIL was 6-fold more than in mice expressing GFP. This effect was reversed (70%) by injection of anti-V5 Ab a day before LL2 cell injection. In spontaneous metastasis, sDC-HIL-V5-transfected LL2 s.c. tumors produced 7-fold more colonies-lung than GFP-LL2 tumors. Relative location of sDC-HIL-accumulated spots (by anti-V5) to LL2 micrometastases (with CSFE) in lung was studied. Surprisingly, sDC-HIL accumulated at only few CD31+ endothelial cells EC (perivascular), in which LL2 tumors were colocalized. Binding of sDC-HIL to the SVEC line led us to examine if EC-bound sDC-HIL is T cell-suppressive. sDC-HIL-coated SVEC markedly suppressed CD8+ T cell activation (by 80%), which was completely reversed by anti-DC-HIL Ab. We then examined this inhibitory effect in vivo. Mice with sDC-HIL-V5 or GFP tumor received i.v. injection of CFSE-labeled activated CD8+ T cells, and 3 d later, the labeled T cells were assayed by FACS. CD8+ T cells in lung with sDC-HIL expression had significantly less # of proliferating cells than control (12% in sDC-HIL vs. 80% in GFP). Thus, the local accumulation of sDC-HIL onto particular EC makes the microenvironment immunosuppressive, a place that may serve as a pre-metastatic niche.
Down-regulated mir-23a contributes to invasion and metastasis of cutaneous melanoma by promoting autophagy Q Shi, H Wang, W Guo, Y Yang, W Zhang, S Guo, T Zhao, L Liu, Z Jian, Q Luan, L Liu, G Wang, T Gao and C Li Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Since the progression of melanoma from primary stage to advanced stage leads to a precipitous drop in the patients’ survival, the mechanisms underlying this process are of great importance. Recently, autophagy has been implicated in modulating cancer invasion and metastasis, and the level of autophagy has been found dysregulated in melanoma. Here, we reported miR-23a as a novel autophagy-regulating microRNA that played a remarkable role in modulating melanoma invasive and metastatic capacity. We found that serum miR-23a level was significantly down-regulated in melanoma patients and highly correlated with poor clinical outcomes. Notably, tissue miR-23a level was drastically decreased in metastatic melanoma compared with that in primary melanoma and melanocytic nevus. Furthermore, ectopic miR-23a expression prevented the invasion-metastasis cascade of melanoma both in vitro and in vivo, and overexpressed miR-23a suppressed the invasive and migratory property of melanoma by abrogating autophagy through directly targeting ATG12. Specially, attenuated melanoma invasion and migration mediated by overexpressed miR-23a resulted from the inhibition on ROS-HIF1a-RhoE signaling. Finally, we revealed that the down-regulation of miR-23a in metastatic melanoma was caused by RUNX2 in a transcriptional repressionmanner. Taken together, our findings demonstrate that miR-23a can act as a crucial epigenetic repressor of melanoma invasion and metastasis in an autophagy-dependent way, which indicates that miR-23a-mediated autophagy inhibition can be exploited to restrain invasionmetastasis cascade in melanoma treatment.
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CD8 to nearest tumor cell distance predicts disease progression in primary intermediate thickness melanoma B Dyring-Andersen1, J Lin1, M Dyring-Andersen1, JT O’Malley1, J Teague1, A Gehad1, C Hoyt2, PA Vieyra1 and R Clark1 1 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA and 2 PerkinElmer, Hopkinton, MA One of the clinical challenges in melanoma is the early identification of patients with intermediate risk melanomas who will go on to develop progressive disease. Effective combination immune checkpoint therapies are now available that can induce regression of even advanced melanoma. An important next step would be to use these therapies early in patients with primary melanomas at high risk for recurrence. A cohort of 30 primary intermediate risk cutaneous malignant melanomas (Breslow thickness 1-2 mm) were divided in two groups depending on sentinel lymph node biopsy (SLNB) status and subsequently thickness matched, resulting in 9 pairs. For pilot studies, six thickness matched tumors were included, 3 tumors from patients with positive SLNB and 3 tumors from patients with negative SLNB. We studied these primary tumors by multiplexed tyramide signal amplification based staining for CD4, CD8, FoxP3 and S100 expression and DAPI, followed by image deconvolution and automated cell analysis. Patients with a negative SLNB had a greater number of CD8+ T cells in tumors than patients with positive SLNB (202 vs. 98, p¼0.04). The numbers of FoxP3+ regulatory T cells and total CD4+ T cells were not significantly different. In breast cancer, the distance between CD8+ T cells and the nearest tumor cells identified patients who would develop progressive disease. We carried out nearest neighbor analysis on melanoma and found that the CD8+ T cell to nearest tumor cell distance was higher in tumors from patients with positive SLNB compared to patients with negative SLNB (120 vs. 70, p¼0.02). We are in the process of confirming this finding in the primary tumors in our initial cohort, as well as in 43 additional primary tumors with data on 5 year survival. In summary, we find that CD8+ T cell density and, more significantly, CD8+ T cell to tumor cell distance predicts progressive disease in primary intermediate risk melanoma.
S110 Journal of Investigative Dermatology (2016), Volume 136
CRD-BP as a potential prognostic marker and therapeutic target in melanoma YG Xu1, T Kim1,2, V Setaluri1 and V Spiegelman1,3 1 Dermatology, University of Wisconsin, Madison, WI, 2 Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI and 3 Department of Pediatrics, Pennsylvania State University, Hershey, PA CRD-BP is an RNA binding protein that binds to various targets such as mRNAs of bTrCP1, c-myc, Gli1, MDR1 and MITF, and affects their translation, localization and stability leading to increased survival, inhibition of apoptosis and development of drug resistance as well as induction of tumorigenic properties in a number of malignancies including melanoma. Current clinical staging of melanoma has missed a subset of thin melanomas that would progress aggressively and even cause death. Existing targeted molecular therapy such as inhibitors of V600E-mutant BRAF and combined inhibitors of BRAF and MARK is hampered by a short median length of response of only about 6 months, resistance to treatments, and no significant impact on overall survival. To explore the prognostic and therapeutic potential of CRD-BP, we have assessed the correlation between elevated CRD-BP expression and melanoma aggressiveness and clinical outcome using the melanoma tumor microarray and the prognostic and diagnostic significance of CRD BP over-expression in melanoma is discussed. Moreover, we have previously reported that inhibition of CRD-BP suppress melanoma growth regardless of BRAF mutation status. We now further demonstrate that in BRAFV600E melanoma cells sensitive to BRAF inhibitor, dual inhibition of mutant BRAF and CRD-BP is significantly more effective than single treatment (p<0.01). In cells with acquired or intrinsic resistance, dual inhibition no longer has additional benefit but down-regulation of CRD-BP alone still significantly inhibits cell growth (p<0.01). Similar results are seen in shortterm melanoma cultures freshly grown from patients, which are biologically more close to human melanoma than established cell lines. In BRAF-mutant melanomas, CRD-BP may be an effective adjuvant therapy to BRAF inhibitors and a novel strategy when resistance develops.