Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu¹²⁸⁰ separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.     

TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.     

BNIP3 promotes HIF‐1α‐driven melanoma growth by curbing intracellular iron homeostasis

BNIP3 is a mitophagy receptor with context‐dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient’s survival and depletion of BNIP3 in B16‐F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2‐mediated downregulation of HIF‐1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3‐deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4‐mediated ferritinophagy, which fostered PHD2‐mediated HIF‐1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF‐1α levels in BNIP3‐depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF‐1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro‐tumorigenic HIF‐1α glycolytic program in melanoma cells.     

Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless αvβ3-Integrin Is Absent

Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial β3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in β3-null mice are all Rac1-dependent. These data indicate that in the presence of αvβ3-integrin Rac1 is not required for tumor angiogenesis.     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

PIASxα Ligase Enhances SUMO1 Modification of PTEN Protein as a SUMO E3 Ligase

The tumor suppressor PTEN plays a critical role in the regulation of multiple cellular processes that include survival, cell cycle, proliferation, and apoptosis. PTEN is frequently mutated or deleted in various human cancer cells to promote tumorigenesis. PTEN is regulated by SUMOylation, but the SUMO E3 ligase involved in the SUMOylation of PTEN remains unclear. Here, we demonstrated that PIASxα is a SUMO E3 ligase for PTEN. PIASxα physically interacted with PTEN both in vitro and in vivo. Their interaction depended on the integrity of phosphatase and C2 domains of PTEN and the region of PIASxα comprising residues 134–347. PIASxα enhanced PTEN protein stability by reducing PTEN ubiquitination, whereas the mutation of PTEN SUMO1 conjugation sites neutralized the effect of PIASxα on PTEN protein half-life. Functionally, PIASxα, as a potential tumor suppressor, negatively regulated the PI3K-Akt pathway through stabilizing PTEN protein. Overexpression of PIASxα led to G₀/G₁ cell cycle arrest, thus triggering cell proliferation inhibition and tumor suppression, whereas PIASxα knockdown or deficiency in catalytic activity abolished the inhibition. Together our studies suggest that PIASxα is a novel SUMO E3 ligase for PTEN, and it positively regulates PTEN protein level in tumor suppression.     

Characterization of Human γδ T Lymphocytes Infiltrating Primary Malignant Melanomas

T lymphocytes are often induced naturally in melanoma patients and infiltrate tumors. Given that γδ T cells mediate antigen-specific killing of tumor cells, we studied the representation and the in vitro cytokine production and cytotoxic activity of tumor infiltrating γδ T cells from 74 patients with primary melanoma. We found that γδ T cells represent the major lymphocyte population infiltrating melanoma, and both Vδ1⁺ and Vδ2⁺ cells are involved. The majority of melanoma-infiltrating γδ cells showed effector memory and terminally-differentiated phenotypes and, accordingly, polyclonal γδ T cell lines obtained from tumor-infiltrating immune cells produced IFN-γ and TNF-α and were capable of killing melanoma cell lines in vitro. The cytotoxic capability of Vδ2 cell lines was further improved by pre-treatment of tumor target cells with zoledronate. Moreover, higher rate of γδ T cells isolation and percentages of Vδ2 cells correlate with early stage of development of melanoma and absence of metastasis. Altogether, our results suggest that a natural immune response mediated by γδ T lymphocytes may contribute to the immunosurveillance of melanoma.     

δ-Catenin Activates Rho GTPase,Promotes Lymphangiogenesis and Growth of Tumor Metastases

δ-catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis.     

EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRIN''s expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα⁺. Germinating spores carrying either a deletion of polα⁺ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.