TIM-4 glycoprotein-mediated degradation of dying tumor cells by autophagy leads to reduced antigen presentation and increased immune tolerance.

Phagocytosis of apoptotic cells by myeloid cells has been implicated in the maintenance of immune homeostasis. In this study, we found that T cell immunoglobulin- and mucin domain-containing molecule-4 (TIM-4) repressed tumor-specific immunity triggered by chemotherapy-induced tumor cell death. TIM-4 was found to be highly expressed on tumor-associated myeloid cells such as macrophages (TAMs) and dendritic cells (TADCs) and danger-associated molecular patterns (DAMPs) released from chemotherapy-damaged tumor cells induced TIM-4 on tumor-associated myeloid cells recruited from bone marrow-derived precursors. TIM-4 directly interacted with AMPKα1 and activated autophagy-mediated degradation of ingested tumors, leading to reduced antigen presentation and impaired CTL responses. Consistently, blockade of the TIM-4-AMPKα1-autophagy pathway augmented the antitumor effect of chemotherapeutics by enhancing tumor-specific CTL responses. Our finding provides insight into the immune tolerance mediated by phagocytosis of dying cells, and targeting of the TIM-4-AMPKα1 interaction constitutes a unique strategy for augmenting antitumor immunity and improving cancer chemotherapy.

Arsenic trioxide prevents osteosarcoma growth by inhibition of GLI transcription via DNA damage accumulation.

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas.

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8(+) T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.

RBPJ is a novel target for rhabdomyosarcoma therapy.

The Notch pathway regulates a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal development. In addition, the Notch pathway plays an important role in controlling tumorigenesis. However, the role of RBPJ, a transcription factor in the Notch pathway, in the development of tumors is largely unknown. In this study, we focused on the role of RBPJ in the pathogenesis of rhabdomyosarcoma (RMS). Our data showed that Notch pathway genes were upregulated and activated in human RMS cell lines and patient samples. Inhibition of the Notch pathway by a γ-secretase inhibitor (GSI) decreased the in vitro proliferation of RMS cells. Knockdown of RBPJ expression by RNAi inhibited the anchorage-independent growth of RMS cells and the growth of xenografts in vivo. Additionally, overexpression of RBPJ promoted the anchorage-independent growth of RMS cells. Further, we revealed that RBPJ regulated the cell cycle in RMS xenograft tumors and decreased proliferation. Our findings suggest that RBPJ regulates the RMS growth, and that the inhibition of RBPJ may be an effective therapeutic approach for patients with RMS.

Role of GOLPH3 and GOLPH3L in the proliferation of human rhabdomyosarcoma.

GOLPH3 was originally identified by proteomic analyses of Golgi proteins localized in the trans-Golgi network. Recently, it was reported that GOLPH3 is up-regulated in various types of malignancies, including melanoma, colon cancer and lung cancer. However, the mechanism through which GOLPH3 is involved in the pathogenesis of rhabdomyosarcoma remains unidentified. In order to explore the function of GOLPH3 and its isoform, GOLPH3L, in the pathogenesis of rhabdomyosarcoma, we investigated the expression and knockdown effects of GOLPH3 and GOLPH3L in human rhabdomyosarcoma. Western blot analysis and real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens exhibited an increased expression of GOLPH3 and GOLPH3L. GOLPH3 and GOLPH3L knockdown by siRNA prevented the proliferation of human rhabdomyosarcoma cell lines. In addition, double-knockdown of GOLPH3 and GOLPH3L also prevented the proliferation of rhabdomyosarcoma cell lines. Our findings improve the understanding of rhabdomyosarcoma pathogenesis and suggest that the knockdown of GOLPH3 or GOLPH3L may be an effective treatment for rhabdomyosarcoma.

Pharmacological inhibition of the Hedgehog pathway prevents human rhabdomyosarcoma cell growth.

The Hedgehog pathway functions as an organizer in embryonic development. Recent studies have shown that mutation of the PTCH1 gene involved in the Hedgehog pathway affects rhabdomyosarcoma development. However, the expression of Hedgehog pathway molecules in human rhabdomyosarcoma cells has not been well clarified. In addition, the effect of pharmacological inhibition of the Hedgehog pathway is not known. We investigated the expression of the genes involved in the Hedgehog pathway using human rhabdomyosarcoma cell lines and biopsy specimens. Further, we evaluated the effect of pharmacological inhibition of the Hedgehog pathway using cyclopamine or GANT61 by WST assay, cell proliferation assay and cell death detection assay. Real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens overexpressed the following genes: Sonic hedgehog, Indian hedgehog, Desert hedgehog, PTCH1, SMO, GLI1, GLI2 and ULK3. Immunohistochemistry revealed that rhabdomyosarcoma cell lines and biopsy specimens expressed SMO and GLI2. Inhibition of SMO by cyclopamine slowed the growth of human rhabdomyosarcoma cell lines. Similarly, inhibition of GLI by GANT61 slowed the growth of human rhabdomyosarcoma cell lines. Inhibition of cell proliferation and apoptotic cell death together prevented the growth of rhabdomyosarcoma cells by cyclopamine and GANT61 treatment. Our findings suggest that pharmacological inhibition of the Hedgehog pathway may be a useful approach for treating rhabdomyosarcoma patients.

Role of GLI2 in the growth of human osteosarcoma.

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real-time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over-expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage-independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G(1) phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21(cip1) . In addition, over-expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma.

Suppression of osteosarcoma cell invasion by chemotherapy is mediated by urokinase plasminogen activator activity via up-regulation of EGR1.

BACKGROUND: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. METHODS: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. RESULTS: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients' biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient' specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted down-regulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. CONCLUSIONS: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods.

Smoothened as a new therapeutic target for human osteosarcoma.

BACKGROUND: The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition. RESULTS: To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo. CONCLUSIONS: These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.