Triptolide Reduces MDA-MB-231 Cell Metastasis by Attenuating Epithelial-Mesenchymal Transition through the ROCK/PTEN/Akt Axis.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and invasive subtype of breast cancer. The prognosis of TNBC is poor because of its high distant metastasis rate. Triptolide is a type of diterpene trioxide natural compound with potential anti-tumor activities. This study explored the metastatic inhibitory effect of triptolide on MDA-MB-231 cells and its underlying mechanism. Triptolide suppressed cell proliferation and induced cell apoptosis in a time- and dose-dependent manner. Low doses of triptolide (0-8 nM) reduced the migration and invasion capabilities of MDA-MB-231 cells. Triptolide decreased ROCK1, p-Akt, N-cadherin, vimentin and MMP-9 expressions, but increased PTEN and E-cadherin expressions on protein and mRNA levels. Furthermore, the down-regulation of ROCK1 expression in MDA-MB-231 cells after being treated by triptolide could be rescued by ROCK1 specific inhibitor Y27632. Molecular docking showed that triptolide and Y27632 shared the same active center of ROCK1 protein. This article's findings taken together showed that ROCK1 is the primary target of triptolide, which can cause cell apoptosis and inhibit the epithelial-mesenchymal transition of MDA-MB-231 cells.

Cisplatin-activated ERß/DCAF8 positive feedback loop induces chemoresistance in non-small cell lung cancer via PTEN/Akt axis.

High levels of the estrogen receptor ß (ERß) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERß remains elusive. In this study, we demonstrated that targeting ERß could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERß, which facilitates its homodimerization and nuclear translocation. ERß activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERß, leading to ERß accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERß are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERß-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.

PTEN mutation predicts unfavorable fertility preserving treatment outcome in the young patients with endometrioid endometrial cancer and atypical hyperplasia.

OBJECTIVE: This study aimed to investigate the impact of molecular classification and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment in the patients with endometrioid endometrial cancer (EEC) and endometrial atypical hyperplasia (EAH). METHODS: This is a single-center retrospective study. A total of 135 patients with EEC and EAH receiving fertility-preserving treatment and molecular classification were reviewed. The distribution of the four types of molecular classification was described. The impact of non-specific molecular profile (NSMP), mismatch repair-deficiency (MMRd), and PTEN, KRAS and PIK3CA gene mutation on the outcome of fertility-preserving treatment was analyzed. RESULTS: Of the patients analyzed, 86.7% (117/136) were classified as having NSMP; 14 (10.4%), MMRd; 1 (0.7%), POLEmut EAH; and 3 (2.2%), p53abn EEC. The patients having NSMP and MMRd achieved similar 16-, 32-, and 48-week complete response rates. The patients harboring tier I and tier II PTEN mutations (PTENmut-Clin) achieved lower cumulative 32-week CR rates than those with PTEN-others (without PTENmut-Clin) (22/47, 46.8% vs. 50/74, 67.6%; p=0.023; odds ratio=0.422; 95% confidence interval [CI]=0.199-0.896). Insulin-resistance (hazard ratio [HR]=0.435; 95% CI=0.269-0.702; p=0.001) and PTENmut-Clin (HR=0.535; 95% CI=0.324-0.885; p=0.015) were independent negative predictors for lower 32-week CR rates. CONCLUSION: PTENmut-Clin is an independent risk factor for unfavorable fertility-preserving treatment outcomes in the patients with EEC and EAH. The patients with MMRd receiving fertility-preserving treatment achieved outcomes similar to those of the patients with NSMP. The molecular profiles might guide fertility-preserving treatment in the prognosis and clinical decisions.

PTEN rs701848 Polymorphism is Associated with Trastuzumab Resistance in HER2-positive Metastatic Breast Cancer and Predicts Progression-free Survival.

BACKGROUND: Trastuzumab is an effective therapeutic approach for HER2-positive metastatic breast cancer (BC). However, a considerable number of patients develop resistance along the course of the disease. PTEN rs701848 polymorphisms are associated with an increased risk of developing cancer and have a potential role in predicting drug resistance. OBJECTIVE: We studied the significance of PTEN rs701848 variants as significant predictors for trastuzumab resistance in HER2-positive metastatic BC patients. Therefore, considering their value in predicting clinical outcomes. MATERIALS AND METHODS: This case-control study was conducted among female patients with HER2-positive metastatic breast cancer who underwent Trastuzumab therapy during the period from March 2017 to December 2020. PTEN rs701848 genotypes were analyzed in 160 HER2-positive metastatic breast cancer who received Trastuzumab therapy and clinically monitored for therapeutic response. RESULTS: PTEN rs701848 is deemed a significant predictor of Trastuzumab resistance and an independent prognostic factor of progression-free survival (PPFS). In particular, the C allele is associated with increased risk for Trastuzumab resistance and shorter PFS as compared to the homozygous TT genotype. CONCLUSION: PTEN rs701848 is significant predictor of trastuzumab resistance. Therefore, their value in predicting clinical outcomes is recommended.

A Phase I Clinical Trial of Trametinib in Combination with TAS-102 in Patients with Chemotherapy-Resistant RAS-Mutated (PIK3CA/PTEN-Wild Type) Metastatic Colorectal Cancer.

PURPOSE: We conducted a phase I study to evaluate the maximum tolerated dose (MTD), safety, and efficacy of trametinib in combination with TAS-102 in patients with chemotherapy-refractory KRAS-mutant, wild-type PIK3CA/PTEN metastatic colorectal cancer (mCRC). METHODS: A 3+3 dose de-escalation single arm phase I clinical trial was performed in patients with chemorefractory mCRC without priorTAS-102 exposure. Patients received fixed dosing of trametinib 2mg oral daily along with de-escalating doses of TAS-102 beginning at 35 mg/m2 twice daily on days 1-5 and days 8-12 every 28 days. Primary endpoint was evaluation of MTD. RESULTS: 25 eligible patients were enrolled in this study. During the dose de-escalation phase, no dose-limiting toxicities (DLT) were observed at the full doses of trametinib/TAS-102 and the MTD was determined to be TAS-102 35 mg/m2 orally twice daily on days 1 to5 and days 8 to12 every 28 days with continuous trametinib dosing at 2mg orally daily. No patients achieved a partial or complete response. 5 of 21 evaluable patients (23.81%) achieved a stable disease response. Median PFS was two months (95% confidence interval [CI] 1.70-4.82) while median OS was 7 months (95% CI 6.36-11.48). Treatments were well tolerated with most common grade ≥ 3 adverse events being anemia (20%), neutropenia (12%), leukopenia (8%), diarrhea (8%), rash (4%), and fatigue (4%). CONCLUSIONS: Trametinib in combination with TAS-102 demonstrated a manageable safety profile. However, this combination did not achieve meaningful clinical benefit in patients with RAS-mutated PIK3CA and PTEN wild-type refractory mCRC. CLINICAL TRIAL INFORMATION: NCT03317119.

Preformulation Studies of a Stable PTEN-PDZ Lipopeptide Able to Cross an In Vitro Blood-Brain-Barrier Model as a Potential Therapy for Alzheimer's Disease.

PURPOSE: Amyloid ß (Aß) drives the accumulation of excess Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) at synapses, inducing synaptic depression and perturbing memory. This recruitment of PTEN to synapses in response to Aß drives its interaction with PSD95/Disc large/Zonula occludens-1 (PDZ) proteins and, indeed, we previously showed that an oligo lipopeptide (PTEN-PDZ) capable of blocking such PTEN:PDZ interactions rescues the synaptic and cognitive deficits in a mouse model of Alzheimer's disease. Hence, the PTEN:PDZ interaction appears to be crucial for Aß-induced synaptic and cognitive impairment. Here we have evaluated the feasibility of using PTEN-PDZ lipopeptides based on the human/mouse PTEN C-terminal sequence, testing their stability in biological fluids, their cytotoxicity, their ability to self-assemble and their in vitro blood-brain barrier (BBB) permeability. Myristoyl or Lauryl tails were added to the peptides to enhance their cell permeability. METHODS: Lipopeptides self assembly was assessed using electron microscopy and the thioflavin T assay. Stability studies in mouse plasma (50%), intestinal washing, brain and liver homogenates as well as permeability studies across an all human 2D blood-brain barrier model prepared with human cerebral endothelial cells (hCMEC/D3) and human astrocytes (SC-1800) were undertaken. RESULTS: The mouse lauryl peptide displayed enhanced overall stability in plasma, ensuring a longer half-life in circulation that meant there were larger amounts available for transport across the BBB (Papp0-4h: 6.28 ± 1.85 × 10-6 cm s-1). CONCLUSION: This increased availability, coupled to adequate BBB permeability, makes this peptide a good candidate for therapeutic parenteral (intravenous, intramuscular) administration and nose-to-brain delivery. Graphical Abstract.

Combinatorial therapy with adenoviral-mediated PTEN and a PI3K inhibitor suppresses malignant glioma cell growth in vitro and in vivo by regulating the PI3K/AKT signaling pathway.

PURPOSE: Glioblastoma is a highly invasive and challenging tumor of the central nervous system. The mutation/deletion of the tumor suppressor phosphatase and tensin homolog (PTEN) gene is the main genetic change identified in glioblastomas. PTEN plays a critical role in tumorigenesis and has been shown to be an important therapeutic target. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is commonly used to inhibit glioma cell growth via regulation of the PI3K/AKT signaling pathway. In this study, we examined the growth inhibitory effects of a combinatorial therapy of adenoviral-mediated PTEN (Ad-PTEN) and LY294002 on LN229 and U251 glioma cells in vitro and on tumor xenografts in vivo. METHODS: In vitro, LN229 and U251 glioma cells were treated by combinatorial therapy with Ad-PTEN and LY294002. The growth ability was determined by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Cell invasive ability was analyzed by transwell invasion assay and cell apoptosis analysis via FITC-Annexin V analysis. In vivo, U251 subcutaneous glioblastoma xenograft was used to assay anti-tumor effect of combinatorial therapy with Ad-PTEN and LY294002 by mean volume of tumors, immunohistochemistry and TUNEL method. RESULTS: The combinatorial treatment clearly suppressed cell proliferation, arrested the cell cycle, reduced cell invasion and promoted cell apoptosis compared with the Ad-PTEN or LY294002 treatment alone. The treatment worked by inhibiting the PI3K/AKT pathway. In addition, the growth of U251 glioma xenografts treated with the combination of Ad-PTEN and LY294002 was significantly inhibited compared with those treated with Ad-PTEN or LY294002 alone. CONCLUSIONS: Our data indicated that the combination of Ad-PTEN and LY294002 effectively suppressed the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach for glioma gene therapy that warrants further investigation.

Effect of adenovirus-mediated PTEN gene on ulcerative colitis-associated colorectal cancer.

PURPOSE: This study investigated the expression pattern of PTEN and its effect on carcinogenesis of ulcerative colitis-associated colorectal cancer, leading to insights into the underlying molecular mechanism. METHODS: We established a mouse model of ulcerative colitis-associated colorectal cancer by treating the animals with azoxymethane (AOM) and dextran sulphate sodium (DSS), and investigated the inflammation-dysplasia-carcinoma sequence. Expression patterns of PTEN, p-Akt and Ki-67 were shown by immunohistochemistry; western blotting techniques were used to detect protein expression of PTEN, p-Akt and caspase 3; TUNEL assay was used to measure apoptosis in colon epithelial cells; and colorimetric analysis was able to determine MPO activity in colon tissues. RESULTS: During the inflammation-dysplasia-carcinoma sequence, PTEN expression gradually decreased, while p-Akt expression increased; PTEN and p-Akt levels were negatively correlated. Compared to the AOM-DSS and Ad-0 groups, Ad-PTEN mice had longer colons, fewer tumours (P < 0.01) and smaller tumour sizes (P < 0.05). After injecting Ad-PTEN, expression of p-Akt, Ki-67 and MPO activity decreased dramatically, whereas PTEN increased. The TUNEL assay showed increased apoptotic cells and caspase 3 expression in the Ad-PTEN group. CONCLUSION: PTEN plays an important role in the inflammation-dysplasia-carcinoma sequence and may be a new molecular target in preventing and treating ulcerative colitis-associated colorectal cancer.

Mannan-modified Ad5-PTEN treatment combined with docetaxel improves the therapeutic effect in H22 tumor-bearing mice.

BACKGROUND: It has been reported that the tumor suppressor gene, PTEN, which is inactivated in many malignant tumors, plays an important role in apoptosis, cell cycle arrest, cell migration, and cell spread. For cancer gene therapy, one of the most important problems is low gene transfection efficiency. METHODS: In the present study, to take full advantage of adenovirus in gene expression, we prepared mannan-modified recombinant adenovirus using the PTEN gene (Man-Ad5-PTEN) and investigated the effect of Man-Ad5-PTEN combined with docetaxel (Man-Ad5-PTEN-docetaxel) on tumor growth in a murine model of hepatocellular carcinoma. RESULTS: Man-Ad5-PTEN effectively suppressed tumor growth and induced significant apoptosis of murine H22 hepatoma in vivo. Apoptosis levels in tumor-bearing mice treated with Man-Ad5-PTEN-docetaxel were significantly higher than those in tumor-bearing mice treated with naked Ad5-PTEN, Man-Ad5-PTEN, or docetaxel alone. Treatment with Man-Ad5-PTEN-docetaxel resulted in a significant inhibitory effect in this tumor model. Compared with the controls treated with phosphate-buffered solution, the tumor inhibition rate with naked Ad5-PTEN, docetaxel, Man-Ad5-PTEN, and Man-Ad5-PTEN-docetaxel was 48.69%, 49.98%, 75.88%, and 96.93%, respectively. CONCLUSION: These results suggest that combined treatment with Man-Ad5-PTEN and other chemotherapeutic agents may be a potent adjuvant therapeutic approach for the treatment of hepatocellular carcinoma.

Intranasal delivery of caspase-9 inhibitor reduces caspase-6-dependent axon/neuron loss and improves neurological function after stroke.

Despite extensive research to develop an effective neuroprotective strategy for the treatment of ischemic stroke, therapeutic options remain limited. Although caspase-dependent death is thought to play a prominent role in neuronal injury, direct evidence of active initiator caspases in stroke and the functional relevance of this activity have not previously been shown. Using an unbiased caspase-trapping technique in vivo, we isolated active caspase-9 from ischemic rat brain within 1 h of reperfusion. Pathogenic relevance of active caspase-9 was shown by intranasal delivery of a novel cell membrane-penetrating highly specific inhibitor for active caspase-9 at 4 h postreperfusion (hpr). Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. The temporal and spatial pattern of expression demonstrates that neuronal caspase-9 activity induces caspase-6 activation, mediating axonal loss by 12 hpr followed by neuronal death within 24 hpr. Collectively, these results support selective inhibition of these specific caspases as an effective therapeutic strategy for stroke.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-ras(LA1) mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.

Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts.

Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.