Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.     

A pilot trial assessing the efficacy of paroxetine hydrochloride (Paxil?) in controlling hot flashes in breast cancer survivors

Background:Many breast cancer survivors suffer debilitating hotflashes. Estrogen, the drug of choice in perimenopausal women, is generallynot recommenced to breast cancer survivors. Nonhormonal treatments are mostlydisappointing. Anecdotal reports in our institution suggested that theselective serotonin-reuptake inhibitor, paroxetine hydrochloride, might beefficacious in alleviating hot flashes. Patients and methods:Thirty women with prior breast cancer whowere suffering at least two hot flashes a day entered a single institutionpilot trial to evaluate paroxetine's efficacy in reducing the frequency andseverity of hot flashes. After completing daily diaries for one week on notherapy, the women received open-label paroxetine, 10 mg daily for one week,followed by four weeks of paroxetine, 20 mg daily. The women completedhot-flash daily diaries throughout the study period, and a health-relatedsymptom-assessment questionnaire and a quality-of-life rating scale in thefirst and sixth week of the study. Results:Twenty-seven women completed the six-week study period.The mean reduction of hot flash frequency was 67% (95%confidence interval (95% CI): 56%–79%). The meanreduction in hot flash severity score was 75% (95% CI:66%–85%). There was a statistically significantimprovement in depression, sleep, anxiety, and quality of life scores.Furthermore, 25 (83%) of the study participants chose to continueparoxetine therapy at the end of study. The most common adverse effect wassomnolence, resulting in drug discontinuation in two women, and dose reductionin two women. One woman discontinued drug due to anxiety. Conclusions:Paroxetine hydrochloride is a promising new treatmentfor hot flashes in breast cancer survivors, and warrants further evaluationin a double-blind randomized placebo-controlled trial.     

Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.     

Differential roles for the p101 and p84 regulatory subunits of PI3Kγ in tumor growth and metastasis

Phosphoinositide 3-kinase γ (PI3Kγ) consists of a catalytic subunit p110γ, which forms mutually exclusive dimers with one of the regulatory subunits called p101 and p84/p87(PIKAP). Recently, PI3Kγ emerged as being a potential oncogene because overexpression of the catalytic subunit p110γ or the regulatory subunit p101 leads to oncogenic cellular transformation and malignancy. However, the contribution of the individual subunits to tumor growth and metastasis and the mechanisms involved are not understood. We therefore individually knocked down the PI3Kγ subunits (p84, p101 and p110γ) in MDA-MB-231 cells, which reduced in vitro migration of the cell lines. Knockdown of p110γ or p101 inhibited apoptosis, Akt phosphorylation and lung colonization in SCID mice. Similarly, the knockdown of p110γ and p101 in murine epithelial carcinoma 4T1.2 cells inhibited primary tumor growth and spontaneous metastasis, as well as lung colonization. In contrast, knockdown of p84 in MDA-MB-231 cells enhanced Akt phosphorylation and lung colonization. These findings are the first to implicate differential functions of the two PI3Kγ regulatory subunits in the process of oncogenesis, and indicate that loss of p101 is sufficient to reduce in vivo tumor growth and metastasis to the same extent as that of p110γ.     

Impact of patient ethnicity on the metabolic and immunologic effects of PI3K–mTOR pathway inhibition in patients with solid tumor malignancies

Purpose

Inhibition of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is associated with metabolic and immunologic perturbations that impact drug tolerability. Here, we studied whether PI3 kinase/mTOR pathway inhibitors are associated with greater metabolic impact and decreased tolerability in Asian patients.

Methods

A retrospective analysis was conducted of consecutive patients with advanced malignancies treated on phase 1 trials of PI3K/mTOR inhibitors. Adverse events related to PI3K/mTOR inhibition, fasting plasma glucose (FPG), insulin, and c-peptide levels, hemoglobin A1c (HgbA1c), and T cell subsets were prospectively collected. Mann–Whitney and Chi-square tests were used to compare continuous and categorical variables, respectively, between Asian and Caucasian patients.

Results

A total of 103 patients (31 Asian; 72 Caucasian) were treated consecutively across five clinical trials. Baseline age, gender distribution, and metabolic parameters were comparable with the exception of lower median body mass index (BMI) in Asian patients (23.0 vs. 24.8 kg/m², p = 0.024). There were no differences in drug tolerability, adherence, or duration of therapy. Asian patients experienced a higher incidence of grade ≥2 hyperglycemia (37.5 vs. 18.1 %, p = 0.03), and greater increases in FPG, HgbA1c, and insulin resistance. No differences in incidence or severity of mucositis, rash, or pneumonitis were observed. Drug effects on neutrophils, lymphocytes, and T cell subsets were similar.

Conclusions

PI3K/mTOR inhibitors have greater glycemic impact in Asian patients, despite similar baseline metabolic parameters, comparable dose intensity, and a lower median BMI. Further studies are warranted to explore the mechanisms underlying these differences and optimize dosing in Asian patients.     

A novel small‐molecule activator of procaspase‐3 induces apoptosis in cancer cells and reduces tumor growth in human breast,liver and gallbladder cancer xenografts

Purpose

Procaspase‐3, a proenzyme of apoptotic executioner caspase‐3, is overexpressed in numerous tumors. We aimed to characterize a novel procaspase‐3 activator, WF‐210, which may have potential as an anticancer drug.

Experimental design

The procaspase‐3 activating ability, antitumor efficacy, mechanisms of action, and toxicity profiles of WF‐210 were investigated in vitro and in vivo, using normal cells, cancer cells, and mouse xenograft models. The role of procaspase‐3 in WF‐210‐induced apoptosis was explored by manipulating procaspase‐3 expression in cultured cells.

Results

WF‐210 activated procaspase‐3 with an EC50 of 0.95 μM, less than half that of its mother compound PAC‐1 (2.08 μM). The mechanism involved the chelation of inhibitory zinc ions, subsequently resulting in an auto‐activation of procaspase‐3. WF‐210 was more cytotoxic than PAC‐1 to human cancer cells, but less cytotoxic to normal cells. Cancer cells with high procaspase‐3 expression, like HL‐60 and U‐937, were particularly sensitive. WF‐210‐induced the apoptosis of HL‐60 and U‐937 cells by activating procaspases and promoting proteasome‐dependent degradation of XIAP and Survivin. The level of WF‐210‐induced apoptosis in cultured cells was related to the level of procaspase‐3 expression. Finally, WF‐210 was superior to PAC‐1 in retarding the in vivo growth of breast, liver and gallbladder xenograft tumors which overexpress procaspase‐3, and induced no substantial weight loss or neurotoxicity. WF‐210 and PAC‐1 had no effect on the growth of MCF‐7 xenograft tumors, which do not express procaspase‐3.

Conclusion

We identified WF‐210 as a potent small‐molecule activator of procaspase‐3. The favorable antitumor activity and acceptable toxicity profile of WF‐210 provide a strong rationale for its clinical evaluation in the treatment of tumors with high procaspase‐3 expression.     

Predictive pharmacokinetic–pharmacodynamic modeling of tumor growth after administration of an anti-angiogenic agent, bevacizumab, as single-agent and combination therapy in tumor xenografts

Purpose

Pharmacokinetic–pharmacodynamic (PK–PD) models able to predict the action of anticancer compounds in tumor xenografts have an important impact on drug development. In case of anti-angiogenic compounds, many of the available models show difficulties in their applications, as they are based on a cell kill hypothesis, while these drugs act on the tumor vascularization, without a direct tumor cell kill effect. For this reason, a PK–PD model able to describe the tumor growth modulation following treatment with a cytostatic therapy, as opposed to a cytotoxic treatment, is proposed here.

Methods

Untreated tumor growth was described using an exponential growth phase followed by a linear one. The effect of anti-angiogenic compounds was implemented using an inhibitory effect on the growth function. The model was tested on a number of experiments in tumor-bearing mice given the anti-angiogenic drug bevacizumab either alone or in combination with another investigational compound. Nonlinear regression techniques were used for estimating the model parameters.

Results

The model successfully captured the tumor growth data following different bevacizumab dosing regimens, allowing to estimate experiment-independent parameters. A combination model was also developed under a ‘no-interaction’ assumption to assess the effect of the combination of bevacizumab with a target-oriented agent. The observation of a significant difference between model-predicted and observed tumor growth curves was suggestive of the presence of a pharmacological interaction that was further accommodated into the model.

Conclusions

This approach can be used for optimizing the design of preclinical experiments. With all the inherent limitations, the estimated experiment-independent model parameters can be used to provide useful indications for the single-agent and combination regimens to be explored in the subsequent development phases.     

GSK-3β inhibition promotes cell death,apoptosis, and in vivo tumor growth delay in neuroblastoma Neuro-2A cell line

Neuroblastoma is the most common extracranial solid tumor of childhood. While survival rates are high for localized disease, treatment response remains poor for a subset of patients with large tumors or disseminated disease. Thus, there remains much room for improvement in treatment strategies for this disease. Using in vitro and in vivo systems, we present glycogen synthase kinase-3β (GSK-3β) inhibition as a potential mechanism to treat neuroblastoma. Using the specific GSK-3β inhibitor SB415286, we demonstrate that GSK-3β inhibition decreases the viability of Neuro-2A cells, as determined by cell proliferation assay and clonogenic survival. Moreover, we show that GSK-3β inhibition induces apoptosis in neuroblastoma cells, as determined by Annexin V staining and confirmed with DAPI staining. Using flow cytometry, we are able to demonstrate that SB415286 induces the accumulation of cells in the G2/M phase of the cell cycle. Finally, we show that these in vitro results translate into delayed tumor growth in vivo using a heterotopic tumor model in nude mice treated with SB415286. These findings suggest that GSK-3β is a potential molecular target for the treatment of neuroblastoma.     

The efficacy of combined treatment with recombinant human tumor necrosis factor-α and 5-fluorouracil is dependent on the development of capillaries in tumor

The antitumor effects of recombinant/human tumor necrosis factor-α (rTNF-α) and 5-fluorouracil (5-FU) in combination treatment were examined on Meth A fibrosarcoma implanted intradermally in mice. Growth of the tumor was inhibited when rTNF-α was given i.v. on day 7 or 11 after implantation, but the effect was countered when 5-FU was additionally given i.p. once a day on days 1–4 after implantation. Conversely, 5-FU given on days 5–8 after implantation augmented the antitumor effects of rTNF-α. Injection of carbon particles showed that fine capillaries did not develop in the tumors of mice treated with 5-FU on days 1–4 after implantation, but that a delicate network of capillaries developed in the tumors of both the mice treated with 5-FU on days 5–8 after implantation and the controls given saline. The results show that the timing of 5-FU treatment is important when attempting to enhance the antitumor effects of rTNF-α, and suggest that these effects are directly associated with newly formed fine capillaries in the tumor.     

Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT

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A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.     

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction  

The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.     

Novel radiosensitizers for locally advanced epithelial tumors: inhibition of the PI3K/Akt survival pathway in tumor cells and in tumor-associated endothelial cells as a novel treatment strategy?

In locally advanced epithelial malignancies, local control can be achieved with high doses of radiotherapy (RT). Concurrent chemoradiotherapy can improve tumor control in selected solid epithelial adult tumors; however, treatment-related toxicity is of major concern and the therapeutic window often small. Therefore, novel pharmacologic radiosensitizers with a tumor-specific molecular target and a broad therapeutic window are attractive. Because of clonal heterogeneity and the high mutation rate of these tumors, combined treatment with single molecular target radiosensitizers and RT are unlikely to improve sustained local tumor control substantially. Therefore, radiosensitizers modulating entire tumor cell survival pathways in epithelial tumors are of potential clinical use. We discuss the preclinical efficacy and the mechanism of three different, potential radiosensitizers targeting the PTEN/PI3K/Akt survival pathway. These compounds were initially thought to act as single-target agents against growth factor receptors (PKI 166 and PTK 787) or protein kinase C isoforms (PKC 412). We describe an additional target for these compounds. PKI 166 (an epidermal growth factor [EGF] receptor inhibitor) and PKC 412, target the PTEN/PI3K/Akt pathway mainly in tumor cells, and PTK 787 (a vascular endothelial growth factor [VEGF] receptor inhibitor) in endothelial cells. Even for these broader range molecular radiosensitizers, the benefit could be restricted to human epithelial tumor cell clones with a distinct molecular profile. Therefore, these potential radiosensitizers have to be carefully tested in specific model systems before introduction in early clinical trials.     

The p85α regulatory subunit of PI3K mediates cAMP-PKA and retinoic acid biological effects on MCF7 cell growth and migration

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.     

HIF-1α siRNA and cisplatin in combination suppress tumor growth in a nude mice model of esophageal squamous cell carcinoma

Introduction: The esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances, and a current challenge is the development of effective therapeutic agents. Our present work addressed the effect of HIF-1α siRNA alone or in combination with cisplatin on the growth of ESCC in nude mice. Materials and Methods: Xenografts were established by inoculating ESCC TE-1 cells in nude mice, and transplanted tumors were treated with HIF-1α siRNA, cisplatin alone or together. Growth was assessed by measuring tumor volume. HIF-1α mRNA and protein expression were detected using RT-PCR and immunohistochemistry, respectively. Apoptosis of ESCC TE-1 cells was analyzed by flow cytometry. Results: In our nude mice model, HIF-1α siRNA effectively inhibited the growth of transplanted ESCC, downregulating HIF-1α mRNA and protein expression, and inducing ESCC TE-1 cell apoptosis. Notably when combinated with cisplatin, HIF-1α siRNA showed synergistic interaction in suppressing tumor growth. Furthermore, the proportion of apoptotic cells in HIF-1α siRNA plus cisplatin group was significantly higher than that in cisplatin or HIF-1α siRNA-treated groups (P<0.05). Conclusions: Down-regulated HIF-1α expression induced by siRNA could effectively suppress the growth of transplanted ESCC in vivo. HIF-1α siRNA could enhance the cytotoxicity of cisplatin, which suggests that a combination of these two agents may have potential for therapy of advanced ESCC.