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Extracellular pH in microenvironment affects anti-tumor activity of inhibitors of mitochondrial respiration against melanoma
Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
prognosis of MF/SS. Here we examined the expression of chemokine receptors in a distinct T cell subpopulation in peripheral blood from a patient with MF through flow cytometry-based segregation. Peripheral blood mononuclear cells were obtained from a patient with erythrodermic MF, and cell surface markers were studied by flow cytometry. We sorted the cells and investigated their morphology, clonality of T-cell receptor rearrangement, and expression of chemokine receptors. CD3+ /CD4+ T cells from the patient’s peripheral blood were separated into two subpopulations; one exhibiting slightly decreased level of CD4 (CD4dim ), the other with obviously lower level of CD4 (CD4dull ) than those in healthy controls. Both CD4dim and CD4dull subpopulations included large lymphocytes with cleaved nuclei, and demonstrated a clonal T-cell receptor rearrangement by PCR. CD26- cells were observed around 70% in the both two subpopulations; however, CD7− cells were 82.5% and 3.7% in CD4dull and CD4dim subpopulations, respectively. Real-time PCR analysis revealed that CD4dull subpopulation demonstrated increased levels of CCR4, CCR7 and CXCR4 by three fold compared with those in CD4dim subpopulation or healthy controls. As reported, malignant nature of lymphoma cells might be associated with aberrant surface phenotype, by which we could distinguish them from normal to less malignant CD4+ T cells. In addition, they preferentially exhibited chemokine receptors CCR4, CCR7 and CXCR4, which might underlie skin infiltration and could be therapeutic targets. http://dx.doi.org/10.1016/j.jdermsci.2016.08.078 P02-11 Extracellular pH in microenvironment affects anti-tumor activity of inhibitors of mitochondrial respiration against melanoma
P02-12 Heat shock factor 1 is required for migration and invasion of human melanoma in vitro and in vivo Yoshitaka Nakamura ∗ , Sonoko Fukushima, Akiko Nakamura, Masahiko Muto Department of Dermatology, Yamaguchi University Graduate School of Medicine, Ube, Japan Heat shock factor 1 (HSF1) is a major transactivator of the heat shock response. Recent studies have demonstrated that HSF1 is involved in tumor initiation, maintenance, and progression by regulating the expression of heat shock proteins (HSPs) and other molecular targets. Furthermore, HSF1 was identified as a potent proinvasion oncogene in human melanomas. However, the biological functions of HSF1 in human melanoma remain poorly understood. To determine the functional role of HSF1 in melanoma, we used short hairpin RNA (shRNA) to silence HSF1 in human melanoma cell lines and investigated its effect on cell migration and invasive ability in vitro. We found that HSF1 knockdown led to a marked reduction in migration and invasive ability, and these functions were restored by overexpression of wild-type HSF1. To confirm the in vitro results, we performed subcutaneous xenograft experiments in athymic nude mice. We found that HSF1 was required for melanoma invasion and metastasis, as well as tumorigenic potential in vivo. Overall, these results show that HSF1 is indispensable for melanoma progression and metastasis, and suggests that HSF1 could be a promising therapeutic target for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.080
Fumihito Noguchi
P02-13
Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan
Cancer testis antigens are expressed in invasive squamous cell carcinoma
Extracellular acidity is a hallmark of cancers. As it contributes to cancer cells acquisition of various malignant phenotypes independent of hypoxia, therapeutic strategies that target cancer cells in acidic microenvironment harbor rationality. In the present study, we revealed that the drugs which abrogate mitochondrial respiration, such as oligomycin and phenformin exhibited enhanced cytotoxicity against mouse and human melanoma cell lines in acidic condition, inhibiting proliferation, attachment and invasion in vitro. Phosphorylation of Akt in the cells was significantly decreased after treated with these drugs in acidic condition, while ATP depletion and reactive oxygen species (ROS) production in the cells partially related to Akt phosphorylation status. The expression of ATP synthase, PGC1alpha and PFKFB3 as well as lactate production after acidification contradicted mitochondorial addiction of the cells. Moreover, MITF-high population, which has been reported to be addicted to mitochondrial respiration, did not decrease in these cell lines after treatment of oligomycin or phenformin at acidic extracellular pH, indicating that the enhanced sensitivity to these drugs is not attributable to mitochondrial addiction of the cells. Furthermore, we demonstrated that intraperitonial injection of phenformin strikingly inhibited lung metastasis of B16F10 in vivo while systemic buffering by sodium bicarbonate rescued both lung metastasis and subcutaneous tumor growth of B16F10. Importantly, these results lead to a notion that the combination of the drugs which inhibit mitochondrial respiration and alkalizer therapy may favor melanoma progression and thus should be avoided in patients with melanoma.
Hiroshi Mitsui 1,2,∗ , Lauren M. Taylor 3 , Mayte ˜ 2 , Kejal R. Shah 4 , Diane Felsen 5 , Suárez-Farinas Shinji Shimada 1 , James G. Krueger 2 , John A. Carucci 3
http://dx.doi.org/10.1016/j.jdermsci.2016.08.079
e23
1
Department of Dermatology, Faculty of Medicine, University of Yamanashi, Japan 2 Laboratory for Investigative Dermatology, the Rockefeller University, USA 3 Ronald O. Perelman Department of Dermatology, New York University Langone Medical Center, USA 4 Texas Dermatology Associates, Baylor University Medical Center, USA 5 Department of Urology, Weill Cornell Medical College, USA Cancer testis antigens (CTAs) are tumor-associated antigens selectively expressed in human tumors, but not in normal tissues except for testis and placenta. CTAs possess strong immunogenicity, thus can be suitable molecular targets for cancer immune therapy. Squamous cell carcinoma (SCC) is a second most common skin cancer. Although many cases are curable by surgical treatment, recent studies have shown that the death rate for SCC approaches that for melanoma in parts of the United States. By combining laser capture microdissection and cDNA microarray techniques, we have previously established gene expression profiling of three distinct transformed keratinocytic regions (actinic keratosis, in situ SCC, and invasive SCC) as well as normal epidermis. We found that CTAs such as, MAGEA3, MAGEA4, and MAGEA6 were selectively
prognosis of MF/SS. Here we examined the expression of chemokine receptors in a distinct T cell subpopulation in peripheral blood from a patient with MF through flow cytometry-based segregation. Peripheral blood mononuclear cells were obtained from a patient with erythrodermic MF, and cell surface markers were studied by flow cytometry. We sorted the cells and investigated their morphology, clonality of T-cell receptor rearrangement, and expression of chemokine receptors. CD3+ /CD4+ T cells from the patient’s peripheral blood were separated into two subpopulations; one exhibiting slightly decreased level of CD4 (CD4dim ), the other with obviously lower level of CD4 (CD4dull ) than those in healthy controls. Both CD4dim and CD4dull subpopulations included large lymphocytes with cleaved nuclei, and demonstrated a clonal T-cell receptor rearrangement by PCR. CD26- cells were observed around 70% in the both two subpopulations; however, CD7− cells were 82.5% and 3.7% in CD4dull and CD4dim subpopulations, respectively. Real-time PCR analysis revealed that CD4dull subpopulation demonstrated increased levels of CCR4, CCR7 and CXCR4 by three fold compared with those in CD4dim subpopulation or healthy controls. As reported, malignant nature of lymphoma cells might be associated with aberrant surface phenotype, by which we could distinguish them from normal to less malignant CD4+ T cells. In addition, they preferentially exhibited chemokine receptors CCR4, CCR7 and CXCR4, which might underlie skin infiltration and could be therapeutic targets. http://dx.doi.org/10.1016/j.jdermsci.2016.08.078 P02-11 Extracellular pH in microenvironment affects anti-tumor activity of inhibitors of mitochondrial respiration against melanoma
P02-12 Heat shock factor 1 is required for migration and invasion of human melanoma in vitro and in vivo Yoshitaka Nakamura ∗ , Sonoko Fukushima, Akiko Nakamura, Masahiko Muto Department of Dermatology, Yamaguchi University Graduate School of Medicine, Ube, Japan Heat shock factor 1 (HSF1) is a major transactivator of the heat shock response. Recent studies have demonstrated that HSF1 is involved in tumor initiation, maintenance, and progression by regulating the expression of heat shock proteins (HSPs) and other molecular targets. Furthermore, HSF1 was identified as a potent proinvasion oncogene in human melanomas. However, the biological functions of HSF1 in human melanoma remain poorly understood. To determine the functional role of HSF1 in melanoma, we used short hairpin RNA (shRNA) to silence HSF1 in human melanoma cell lines and investigated its effect on cell migration and invasive ability in vitro. We found that HSF1 knockdown led to a marked reduction in migration and invasive ability, and these functions were restored by overexpression of wild-type HSF1. To confirm the in vitro results, we performed subcutaneous xenograft experiments in athymic nude mice. We found that HSF1 was required for melanoma invasion and metastasis, as well as tumorigenic potential in vivo. Overall, these results show that HSF1 is indispensable for melanoma progression and metastasis, and suggests that HSF1 could be a promising therapeutic target for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.080
Fumihito Noguchi
P02-13
Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan
Cancer testis antigens are expressed in invasive squamous cell carcinoma
Extracellular acidity is a hallmark of cancers. As it contributes to cancer cells acquisition of various malignant phenotypes independent of hypoxia, therapeutic strategies that target cancer cells in acidic microenvironment harbor rationality. In the present study, we revealed that the drugs which abrogate mitochondrial respiration, such as oligomycin and phenformin exhibited enhanced cytotoxicity against mouse and human melanoma cell lines in acidic condition, inhibiting proliferation, attachment and invasion in vitro. Phosphorylation of Akt in the cells was significantly decreased after treated with these drugs in acidic condition, while ATP depletion and reactive oxygen species (ROS) production in the cells partially related to Akt phosphorylation status. The expression of ATP synthase, PGC1alpha and PFKFB3 as well as lactate production after acidification contradicted mitochondorial addiction of the cells. Moreover, MITF-high population, which has been reported to be addicted to mitochondrial respiration, did not decrease in these cell lines after treatment of oligomycin or phenformin at acidic extracellular pH, indicating that the enhanced sensitivity to these drugs is not attributable to mitochondrial addiction of the cells. Furthermore, we demonstrated that intraperitonial injection of phenformin strikingly inhibited lung metastasis of B16F10 in vivo while systemic buffering by sodium bicarbonate rescued both lung metastasis and subcutaneous tumor growth of B16F10. Importantly, these results lead to a notion that the combination of the drugs which inhibit mitochondrial respiration and alkalizer therapy may favor melanoma progression and thus should be avoided in patients with melanoma.
Hiroshi Mitsui 1,2,∗ , Lauren M. Taylor 3 , Mayte ˜ 2 , Kejal R. Shah 4 , Diane Felsen 5 , Suárez-Farinas Shinji Shimada 1 , James G. Krueger 2 , John A. Carucci 3
http://dx.doi.org/10.1016/j.jdermsci.2016.08.079
e23
1
Department of Dermatology, Faculty of Medicine, University of Yamanashi, Japan 2 Laboratory for Investigative Dermatology, the Rockefeller University, USA 3 Ronald O. Perelman Department of Dermatology, New York University Langone Medical Center, USA 4 Texas Dermatology Associates, Baylor University Medical Center, USA 5 Department of Urology, Weill Cornell Medical College, USA Cancer testis antigens (CTAs) are tumor-associated antigens selectively expressed in human tumors, but not in normal tissues except for testis and placenta. CTAs possess strong immunogenicity, thus can be suitable molecular targets for cancer immune therapy. Squamous cell carcinoma (SCC) is a second most common skin cancer. Although many cases are curable by surgical treatment, recent studies have shown that the death rate for SCC approaches that for melanoma in parts of the United States. By combining laser capture microdissection and cDNA microarray techniques, we have previously established gene expression profiling of three distinct transformed keratinocytic regions (actinic keratosis, in situ SCC, and invasive SCC) as well as normal epidermis. We found that CTAs such as, MAGEA3, MAGEA4, and MAGEA6 were selectively