Epi-reevesioside F inhibits Na+/K+-ATPase,causing cytosolic acidification,Bak activation and apoptosis in glioblastoma

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC₅₀ values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na⁺/K⁺-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na⁺ but not Ca²⁺, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na⁺/K⁺-ATPase, leading to overload of intracellular Na⁺ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.     

Na+/K+-ATPase β2-subunit (AMOG) expression abrogates invasion of glioblastoma-derived brain tumor-initiating cells

Background

Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na⁺/K⁺-ATPase, called the adhesion molecule on glia (AMOG), is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype, we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression.

Methods

Immunohistochemistry, immunoblotting, and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples, BTICs, and differentiated cells. Matrigel invasion assay, scratch assay, and direct cell counting were used for testing in vitro invasion, migration, and proliferation, respectively.

Results

While AMOG expression is heterogeneous in astrocytomas of grades II–IV, it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion.

Conclusions

AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation.     

Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death

A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8⁺ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8⁺ T cells, thereby enhancing their role in tumor immunosurveillance.     

End-binding 1 protein overexpression correlates with glioblastoma progression and sensitizes to Vinca-alkaloids in vitro and in vivo

End-binding 1 protein (EB1) is a key player in the regulation of microtubule (MT) dynamics. Here, we investigated the role of EB1 in glioblastoma (GBM) tumor progression and its potential predictive role for response to Vinca-alkaloid chemotherapy. Immunohistological analysis of the 109 human GBM cases revealed that EB1 overexpression correlated with poor outcome including progression-free survival and overall survival. Downregulation of EB1 by shRNA inhibited cell migration and proliferation in vitro. Conversely, EB1 overexpression promoted them and accelerated tumor growth in orthotopically-transplanted nude mice. Furthermore, EB1 was largely overexpressed in stem-like GBM6 that display in vivo a higher tumorigenicity with a more infiltrative pattern of migration than stem-like GBM9. GBM6 showed strong and EB1-dependent migratory potential. The predictive role of EB1 in the response of GBM cells to chemotherapy was investigated. Vinflunine and vincristine increased survival of EB1-overexpressing U87 bearing mice and were more effective to inhibit cell migration and proliferation in EB1-overexpressing clones than in controls. Vinca inhibited the increase of MT growth rate and growth length induced by EB1 overexpression. Altogether, our results show that EB1 expression level has a prognostic value in GBM, and that Vinca-alkaloid chemotherapy could improve the treatment of GBM patients with EB1-overexpressing tumor.     

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

Background

Depletion of calcium (Ca²⁺) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway which sustains long-term Ca²⁺ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca²⁺ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.

Methods

Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.

Results

We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21^Waf1/Cip1_, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.

Conclusion

Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.     

Isolation and characterization of cancer stem like cells in human glioblastoma cell lines

To identify and compare the features of stem like cells in human glioblastoma cell lines U251, U87MG, A172 with primary cultured glioblastoma stem cells, the ratio of CD133⁺ cells, the ability of tumor sphere formation, and self-renewing capacity of U251, U87MG, A172 cells in serum free medium plus EGF, bFGF and B27 supplement were detected. The results suggested that there might be more cancer stem like cells in U251 cells compared with others. CD133⁺ cells enriched in SP cells and in U251 cells cultured with the serum free medium. They expressed the neural stem cell markers CD133 and Nestin, but lacked of neuronal and astrocyte marker MAP2, β-III tubulin and GFAP. They could apparently generate both neurons and glial cells after serum retrieved in vitro. Gli1, Bmi1, Notch2 and PTEN were also found expressed highly in them. Moreover, CD133⁺ cells were more resistant to hypoxia, irradiations and some chemotherapeutics than CD133⁻cells. So we suggested that glioblastoma stem like cells were existed in CD133⁺ cells in U251 cell line with characteristics of self-renew and generation of an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may be exploited in the development of novel cancer therapeutic drugs.     

CD4+ T cells play a crucial role for lenalidomide in vivo anti-tumor activity in murine multiple myeloma

Lenalidomide modulates the host immune response against myeloma via multiple actions. Although these effects have been elucidated in vitro, the central action of lenalidomide-mediated anti-myeloma immune response in vivo is not clear. To investigate its immune action in vivo, we selected the murine myeloma cell line 5TGM1, which is resistant to direct tumoricidal effects of lenalidomide in vitro and in immunodeficient mice, but sensitive to lenalidomide treatment in 5TGM1-bearing immunocompetent mice. Depletion of CD4⁺ T cells, but not NK cells, B cells, or CD8⁺ T cells, deprived lenalidomide of its therapeutic effects on 5TGM1-bearing immunocompetent mice. Lenalidomide significantly increased the numbers of IFN-γ-secreting CD4⁺ and CD8⁺ T cells but had no effects on NK cells and B cells in this mouse model. Lenalidomide slightly decreased the number of CD25⁺Foxp3⁺ T cells but increased perforin expression in CD8⁺ T cells in vivo. Using this mouse model for investigation of anti-tumor immune action of lenalidomide, we demonstrated that lenalidomide facilitated a type-1 anti-tumor immune response in vivo. The CD4⁺ T cell subset may play a critical role in the lenalidomide-mediated anti-myeloma immune response in vivo.     

NAD depletion by APO866 in combination with radiation in a prostate cancer model,results from an in vitro and in vivo study

Background

APO866 is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), inhibition of which reduces cellular NAD⁺ levels. In this study we addressed the potential of NAD⁺ depletion as an anti-cancer strategy and assessed the combination with radiation.

Methods

The anticipated radiosensitizing property of APO866 was investigated in prostate cancer cell lines PC3 and LNCaP in vitro and in PC3 xenografts in vivo.

Results

We show that APO866 treatment leads to NAD⁺ depletion. Combination experiments with radiation lead to a substantial decrease in clonogenic cell survival in PC3 and LNCaP cells.In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD⁺ levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Compared with untreated tumors, APO866 and radiation alone resulted in tumor growth delays of 14 days and 33 days, respectively, whereas the combination showed a significantly increased tumor growth delay of 65 days.

Conclusions

Our studies show that APO866-induced NAD⁺ depletion enhances radiation responses in tumor cell survival in prostate cancer. However, the in vitro data do not reveal a solid cellular mechanism to exploit further clinical development at this moment.     

Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells

CD8⁺ T cell-mediated immune response plays an important role in inhibiting progression of hepatocellular carcinoma (HCC). For strategic immunotherapy, it is critical to understand why some of the tumor cells escape from this immune attack. In this study, we investigated how HCC cells alter endogenous anti-tumor immunity and their related signaling pathways. We found that HCC cells, both in vitro and in vivo, substantially secret and express amphiregulin (AR). AR in turn activates immunosuppressive function of intratumoral CD4⁺Foxp3⁺ regulatory T cells (Tregs), a major inhibitor of CD8⁺ T cells. Using either lentiviral siRNA, or AR neutralizing antibody, we blocked the expression and function of AR to test the specificity of AR mediated activation of Tregs, Biochemical and cell biology studies were followed and confirmed that blocking of AR inhibited Tregs activation. In addition, we found that AR can trigger the activation of rapamycin complex 1(mTORC1) signaling in Tregs. The mTORC1 inhibitor rapamycin treatment led to compromise Treg function and resulted in enhancing anti-tumor function of CD8⁺ T cells. Blocking AR/EGFR signaling in Tregs with Gefitinib also enhanced anti-tumor immunity and decreased tumor size in a mouse xenograft tumor model. Taken together, our study suggested a novel mechanism of functional interaction between HCC and Tregs for regulating anti-tumor function of CD8⁺ T cells.     

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

O⁶-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (χ² test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76–17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45–2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.     

CD4+ T cell exhaustion leads to adoptive transfer therapy failure which can be prevented by immune checkpoint blockade

Cytotoxic CD8⁺ T cell exhaustion is one of the mechanisms underlying the tumor immune escape. The paradigm-shifting immune checkpoint therapy can mitigate CD8⁺ T lymphocyte exhaustion, reinvigorate the anticancer immunity, and achieve durable tumor regression for some patients. Emerging evidence indicates that CD4⁺ T lymphocytes also have a critical role in anticancer immunity, either by directly applying cytotoxicity toward cancer cells or as a helper to augment CD8⁺ T cell cytotoxicity. Whether anticancer CD4⁺ T lymphocytes undergo exhaustion during immunotherapy of solid tumors remains unknown. Here we report that melanoma antigen TRP-1/gp75-specific CD4⁺ T lymphocytes exhibit an exhaustion phenotype after being adoptively transferred into mice bearing large subcutaneous melanoma. Exhaustion of these CD4⁺ T lymphocytes is accompanied with reduced cytokine release and increased expression of inhibitory receptors, resulting in loss of tumor control. Importantly, we demonstrate that PD-L1 immune checkpoint blockade can prevent exhaustion, induce proliferation of the CD4⁺ T lymphocytes, and consequently prevent tumor recurrence. Therefore, when encountering an excessive amount of tumor antigens, tumor-reactive CD4⁺ T lymphocytes also enter the exhaustion state, which can be prevented by immune checkpoint blockade. Our results highlight the importance of tumor-specific CD4⁺ T lymphocytes in antitumor immunity and suggest that the current immune checkpoint blockade therapy may achieve durable anticancer efficacy by rejuvenating both tumor antigen-specific CD8⁺ T lymphocytes and CD4⁺ T lymphocytes.     

Expansion of CD3+CD8+PD1+ T lymphocytes and TCR repertoire diversity predict clinical responses to adoptive cell therapy in advanced gastric cancer

The adoptive cell therapy (ACT) and delivery of ex vivo activated cellular products, such as dendritic cells (DCs), NK cells, and T cells, have shown promise for the treatment of gastric cancer (GC). However, it is unknown which cells can improve patient survival. This study was focused on the antitumour activity of a subset of these cellular products and their relationships with clinical outcomes. Nineteen patients were enrolled at the Capital Medical University Cancer Center, Beijing Shijitan Hospital, from June 1, 2013, to May 30, 2016. CD8⁺PD1⁺ T-cell sorting was carried out using flow cytometry, and the T-cell receptor (TCR) repertoire during ex vivo expansion for 15 days was analyzed by next-generation sequencing. After 15 days of culture, the number of CD8⁺ T cells had increased significantly, and the number of CD4⁺ T cells had increased correspondingly. After ex vivo expansion, CD8⁺ T cells exhibited significantly enhanced expression of PD-1, LAG-3, and TIM-3 but not 4-1BB. Survival analysis showed that patients with a pro/pre value of CD8⁺PD-1⁺ T cells >2.4 had significantly favorable overall survival (OS) (median OS time, 248 days versus 96 days, P=0.02) and progression-free survival (PFS) (median PFS time, 183 days vs. 77 days, P=0.002). The sorted CD8⁺PD-1⁺ T cells displayed enhanced antitumor activity and increased IFN-γ secretion after coculture with autologous tumor cell lines. TCR repertoire diversity was decreased after ex vivo expansion, which decreased the Shannon index and increased the clonality value. The prognosis of patients was significantly improved and was associated with the extent of CD8⁺PD-1⁺ T-cell expansion. In summary, this study showed that after ex vivo expansion for 15 days, CD8⁺PD-1⁺ T cells could be identified as tumor-reactive cells in patients treated for GC. Changing TCR species can predict the extent of CD3⁺CD8⁺PD1⁺ T-cell growth and the effect of ACT treatment.     

Characterization and immunotherapeutic potential of γδ T-cells in patients with glioblastoma

Classical immunotherapeutic approaches to glioblastoma multiforme (GBM) have shown mixed results, and therapies focused on innate lymphocyte activity against GBM have not been rigorously evaluated. We examined peripheral blood lymphocyte phenotype, γδ T-cell number, mitogenic response, and cytotoxicity against GBM cell lines and primary tumor explants from GBM patients at selected time points prior to and during GBM therapy. Healthy volunteers served as controls and were grouped by age. T-cell infiltration of tumors from these patients was assessed by staining for CD3 and T-cell receptor γδ. Our findings revealed no differences in counts of mean absolute T-cells, T-cell subsets CD3⁺CD4⁺ and CD3⁺CD8⁺, and natural killer cells from healthy volunteers and patients prior to and immediately after GBM resection. In contrast, γδ T-cell counts and mitogen-stimulated proliferative response of γδ T-cells were markedly decreased prior to GBM resection and throughout therapy. Expanded/activated γδ T-cells from both patients and healthy volunteers kill GBM cell lines D54, U373, and U251, as well as primary GBM, without cytotoxicity to primary astrocyte cultures. Perivascular T-cell accumulation was noted in paraffin sections, but no organized T-cell invasion of the tumor parenchyma was seen. Taken together, these data suggest that γδ T-cell depletion and impaired function occur prior to or concurrent with the growth of the tumor. The significant cytotoxicity of expanded/activated γδ T-cells from both healthy controls and selected patients against primary GBM explants may open a previously unexplored approach to cellular immunotherapy of GBM.     

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c⁺CD8⁺ T cells. The CD11c⁺CD8⁺ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c⁺CD8⁺ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.     

The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion

Objective

This study aimed to assess the prognostic value of CD4⁺CD25⁺ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy.

Methods

217 patients participated in the follow-up study. CD4⁺CD25⁺ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4⁺CD25⁺ proportion more than 5% and less than or equal to 5% in peripheral T cells.

Results

Peripheral CD4⁺CD25⁺ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4⁺CD25⁺ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4⁺CD25⁺proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients.

Conclusion

Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4⁺CD25⁺T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.     

Synergistic antitumor efficacy against the EGFRvIII+HER2+ breast cancers by combining trastuzumab with anti-EGFRvIII antibody CH12

Although Trastuzumab, an anti-HER2 antibody, benefits certain patients with HER2-overexpressing breast cancer, de novo or acquired trastuzumab resistance remains a haunting issue. EGFRvIII, co-expressing with HER2 in some breast tumors, indicates a poor clinical prognosis. However, the role of EGFRvIII in the function of trastuzumab is not clear. Here, we demonstrated that EGFRvIII overexpression contributed to de novo trastuzumab resistance and the feedback activation of STAT3 caused by trastuzumab also resulted in acquired resistance in EGFRvIII⁺HER2⁺ breast cancers. CH12, a highly effective anti-EGFRvIII monoclonal antibody that preferentially binds to EGFRvIII, significantly suppressed the growth of EGFRvIII⁺HER2⁺ breast cancer cells in vitro and in vivo. Importantly, CH12 in combination with trastuzumab had a synergistic inhibitory effect on EGFRvIII⁺HER2⁺ breast cancers in vitro and in vivo via attenuating the phosphorylation of EGFR and HER2 and their downstream signal pathways more effectively and reversing STAT3 feedback activation. Moreover, the combination therapy suppressed angiogenesis and induced cell apoptosis significantly. Together, these results suggested a synergistic efficacy of the combination of trastuzumab with CH12 against EGFRvIII⁺HER2⁺ breast cancers, which might be a potential clinical application in the future.     

Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells

Cyclin-dependent kinase inhibitors (CDKIs) are considered as novel anticancer agents because of their ability to induce growth arrest or apoptosis in tumour cells. It has not yet been fully determined, however, which CDKI is the best candidate for the treatment of malignant gliomas and whether normal brain tissues are affected by CDKI expression. Using recombinant adenoviral vectors that express CDKIs (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)), we compared the antitumour effect of CDKIs on malignant glioma cell lines (A172, GB-1, T98G, U87-MG, U251-MG and U373-MG). p27(KIP1) showed higher ability to suppress the growth of all tumour cells tested than other CDKIs. Interestingly, overexpression of p27(KIP1) induced autophagic cell death, but not apoptosis in tumour cells. On the other hand, p27(KIP1) overexpression did not inhibit the viability of cultured astrocytes (RNB) nor induced autophagy. Overall, our findings suggest that gene transfer of p27(KIP1) may be a promising approach for the therapy of malignant gliomas.     

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.     

A subpopulation of CD133 cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy

The potential role of cancer stem-like cells (CSCs) in chemoresistance of human oral squamous cell carcinoma (OSCC) was examined. A small subpopulation (1–2%) of CD133⁺ CSCs was identified in OSCC cell lines and tissues. These CD133⁺ CSCs possess higher clonogenicity, invasiveness, and increased in vivo tumorigenicity as compared to CD133⁻ counterparts. Meanwhile, CD133⁺ CSCs were substantially resistant to standard chemotherapy, wherein both in vitro and in vivo treatment with paclitaxel resulted in a marked enrichment for CD133⁺ CSCs. Our data suggest that CD133⁺ cells represent a small subpopulation of CSCs that may contribute to chemoresistance in human OSCC.