Sulforaphane (SFN) is a promising cancer prevention and treatment agent that strongly suppresses the cutaneous squamous cell carcinoma (CSCC) cell cancer phenotype. We previously showed that yes-associated protein 1 (YAP1)/TEAD signaling is a key procancer stimulator of the aggressive CSCC cell cancer phenotype. However, SFN-responsive upstream regulators of YAP1/TEAD signaling are not well characterized and so there is a pressing need to identify these factors.We show that CD44v6 knockdown reduces YAP1/TEAD-dependent transcription and target gene expression, and that this is associated with reduced spheroid formation, invasion and migration. CD44v6 knockout cell lines also display reduced YAP1/TEAD activity and target gene expression and attenuated spheroid formation, invasion, migration and tumor formation. An important finding is that SFN treatment suppresses CD44v6 level leading to a reduction in YAP1/TEAD signaling and marker gene expression. Sox2 level and epithelial-mesenchymal transition (EMT) are also reduced. Forced expression of constitutive active YAP1 in CD44v6 knockdown cells partially restores the aggressive cancer phenotype. These important findings suggest that CD44v6 drives YAP1/TEAD signaling to enhance the CSCC cell cancer phenotype and that SFN treatment reduces CD44v6 level/function which, in turn, reduces YAP1/TEAD signaling leading to reduced stemness, EMT and tumor growth.
Transglutaminase 2 (TG2) is an important cancer stem-like cell survival protein that is highly expressed in epidermal squamous cell carcinoma and drives an aggressive cancer phenotype. In the present study, we show that TG2 knockdown or inactivation results in a reduction in mammalian target of rapamycin (mTOR) level and activity in epidermal cancer stem-like cells which are associated with reduced spheroid formation, invasion, and migration, and reduced cancer stem cell and epithelial-mesenchymal transition (EMT) marker expression. Similar changes were observed in both cultured cells and tumors. mTOR knockdown or treatment with rapamycin phenocopies the reduction in spheroid formation, invasion, and migration, and cancer stem cell and EMT marker expression. Moreover, mTOR appears to be a necessary mediator of TG2 action, as a forced expression of constitutively active mTOR in TG2 knockdown cells partially restores the aggressive cancer phenotype and cancer stem cell and EMT marker expression. Tumor studies show that rapamycin reduces tumor growth and cancer stem cell marker expression and EMT.These studies suggest that TG2 stimulates mTOR activity to stimulate cancer cell stemness and EMT and drive aggressive tumor growth.cancer stem cells, epidermal squamous cell carcinoma, mTOR, transglutaminase 2 | INTRODUCTIONTransglutaminase 2 (TG2) is a uniquely oncogenic member of the transglutaminase family that is a focus of intense study relating to its role as a cancer cell survival factor. 1,2 TG2 displays transamidase and GTP binding activity, and recent studies show that GTP binding activity is required to maintain the cancer phenotype. 1-4 TG2 level and signaling activity are markedly increased in tumors and tumor cell lines [5][6][7][8][9] where it suppresses expression of tumor suppressor genes, drives synthesis, and deposition of fibronectin and collagen, stabilizes the extracellular matrix and stimulates epithelial-mesenchymal transition (EMT). 4,9,10 Recent studies indicate that TG2 is highly enriched in epidermal cancer stem-like cells (ECS cells) where it stimulates an aggressive cancer phenotype. [2][3][4]11 It localizes in the extra-and intracellular environment where it regulates a host of procancer plasma membrane receptors and intracellular signaling cascades. 3,[11][12][13][14][15] Mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 comprise two distinct functional complexes. mTORC1 (mammalian target of rapamycin [mTOR]) is involved in driving the survival of cancer cells. 16 mTOR is activated in response to nutrient conditions favorable for cell growth and in response to signaling
Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapyresistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dosedependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy for CSCC.
Glutamine addiction is an important phenotype displayed in some types of cancer. In these cells, glutamine depletion results in a marked reduction in the aggressive cancer phenotype. Mesothelioma is an extremely aggressive disease that lacks effective therapy. In this study, we show that mesothelioma tumors are glutamine addicted suggesting that glutamine depletion may be a potential therapeutic strategy. We show that glutamine restriction, by removing glutamine from the medium or treatment with inhibitors that attenuate glutamine uptake (V-9302) or conversion to glutamate (CB-839), markedly reduces mesothelioma cell proliferation, spheroid formation, invasion, and migration. Inhibition of the SLC1A5 glutamine importer, by knockout or treatment with V-9302, an SLC1A5 inhibitor, also markedly reduces mesothelioma cell tumor growth. A relationship between glutamine utilization and YAP1/TEAD signaling has been demonstrated in other tumor types, and the YAP1/TEAD signaling cascade is active in mesothelioma cells and drives cell survival and proliferation. We therefore assessed the impact of glutamine depletion on YAP1/TEAD signaling. We show that glutamine restriction, SLC1A5 knockdown/ knockout, or treatment with V-9302 or CB-839, reduces YAP1 level, YAP1/TEADdependent transcription, and YAP1/TEAD target protein (e.g., CTGF, cyclin D1, COL1A2, COL3A1, etc.) levels. These changes are observed in both cells and tumors. These findings indicate that mesothelioma is a glutamine addicted cancer, show that glutamine depletion attenuates YAP1/TEAD signaling and tumor growth, and suggest that glutamine restriction may be useful as a mesothelioma treatment strategy.
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