NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations

Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.     

A MEK/PI3K/HDAC inhibitor combination therapy for KRAS mutant pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive, metastatic disease with limited treatment options. Factors contributing to the metastatic predisposition and therapy resistance in pancreatic cancer are not well understood. Here, we used a mouse model of KRAS-driven pancreatic carcinogenesis to define distinct subtypes of PDAC metastasis: epithelial, mesenchymal and quasi-mesenchymal. We examined pro-survival signals in these cells and the therapeutic response differences between them. Our data indicate that the initiation and maintenance of the transformed state are separable, and that KRAS dependency is not a fundamental constant of KRAS-initiated tumors. Moreover, some cancer cells can shuttle between the KRAS dependent (drug-sensitive) and independent (drug-tolerant) states and thus escape extinction. We further demonstrate that inhibition of KRAS signaling alone via co-targeting the MAPK and PI3K pathways fails to induce extensive tumor cell death and, therefore, has limited efficacy against PDAC. However, the addition of histone deacetylase (HDAC) inhibitors greatly improves outcomes, reduces the self-renewal of cancer cells, and blocks cancer metastasis in vivo. Our results suggest that targeting HDACs in combination with KRAS or its effector pathways provides an effective strategy for the treatment of PDAC.     

Anticancer activity of HS-527, a novel inhibitor targeting PI3-kinase in human pancreatic cancer cells

Pancreatic cancer is known to have low 5-year survival rate and poor response to treatment. In this study, we synthesized HS-527, a new PI3-kinase inhibitor, and investigated not only its anticancer activity, but also its mechanism of action in pancreatic cancer cells. HS-527 had higher specificity for PI3K than other kinases and inhibited PI3K/Akt signaling pathway by down-regulating Akt and P70S6K. And HS-527 inhibited the cell growth and proliferation of the pancreatic cancer in a time- and dose-dependent manner, with greater activity than gemcitabine. Even HS-527 showed lower cytotoxicity than gemcitabine in normal cells. When treated with HS-527, the cancer cells appeared apoptotic, increasing the expression of cleaved PARP, cleaved caspase-3, and Bax. Furthermore, HS-527 showed an anti-angiogenic activity by decreasing the expression of HIF-1α and VEGF, and inhibited the migration of endothelial cells, and the formation of new blood vessel in mouse Matrigel plug assay. In this study, we found that HS-527 showed anti-cancer activity through an inhibition of the PI3K/Akt pathway in pancreatic cancer cells, suggesting that HS-527 could be used as a promising therapeutic agent for pancreatic cancer.     

Treatment with the PI3K inhibitor buparlisib (NVP-BKM120) suppresses the growth of established patient-derived GBM xenografts and prolongs survival in nude rats

Glioblastomas (GBMs) are aggressive brain tumours with a dismal prognosis, despite combined surgery, radio- and chemotherapy. Close to 90?% of all GBMs harbour a deregulated PI3K pathway, which is essential in regulating central cellular functions such as proliferation, cell growth, motility and survival. Thus, PI3K represents a potential target for molecular therapy in GBM. We investigated the anti-tumour efficacy of the PI3K inhibitor buparlisib (NVP-BKM120) in GBM cell lines in vitro and in vivo, when treatment was initiated after MRI-confirmed tumour engraftment. We found that buparlisib inhibited glioma cell proliferation in a dose dependent manner, demonstrated by MTS assay, manual cell count and BrdU incorporation. A dose dependent increase in apoptosis was observed through flow cytometric analysis. Furthermore, by immunocytochemistry and western blot, we found a dose dependent inhibition of Akt phosphorylation. Moreover, buparlisib prolonged survival of nude rats harboring human GBM xenografts in three independent studies and reduced the tumours’ volumetric increase, as determined by MRI. In addition, histological analyses of xenograft rat brains showed necrotic areas and change in tumour cell nuclei in buparlisib-treated animals. The rats receiving buparlisib maintained their weight, activity level and food- and water intake. In conclusion, buparlisib effectively inhibits glioma cell proliferation in vitro and growth of human GBM xenografts in nude rats. Moreover, the compound is well tolerated when administered at doses providing anti-tumour efficacy. Thus, buparlisib may have a future role in glioma therapy, and further studies are warranted to validate this compound for human use.     

Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma

The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis, and invasion is found in 70%–80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma. We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866 inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression.     

Antineoplastic effect of a single oral dose of the novel Flt3 inhibitor KRN383 on xenografted human leukemic cells harboring Flt3-activating mutations

Activating mutations of Fms-like tyrosine kinase 3 (Flt3) are the most common genetic abnormalities found in acute myeloid leukemia (AML) and represent potential therapeutic targets. The novel Flt3 inhibitor KRN383 inhibited the autophosphorylation of Flt3 bearing internal tandem duplications (ITDs) and the Asp835Tyr (D835Y) point mutation with half-maximal inhibitory concentration (IC₅₀) values of ≤5.9 and 43 nM, respectively. KRN383 also inhibited the proliferation of the ITD-positive cell lines with IC₅₀ values of ≤2.9 nM. A single oral administration of 80 mg/kg of KRN383 eradicated ITD-positive xenograft tumors in nude mice and prolonged the survival of SCID mice carrying ITD-positive AML cells. The effectiveness of a single oral dose of KRN383 suggests that it has the potential to be used in a wide variety of clinical regimens, including multicycle and combination therapies.     

红花多糖抑制PI3K/Akt信号通路诱导人胃癌细胞凋亡的研究

目的 探讨红花多糖通过抑制PI3K/Akt信号通路调控胃癌细胞凋亡的机制。方法 MTT比色法观察SPS对人胃癌SGC-7901细胞体外增殖的抑制作用,流式细胞仪(FCM)分析细胞凋亡,实时荧光定量RT-PCR法和Western Blot法检测蛋白激酶B(Akt)基因及蛋白的表达情况。结果 红花多糖在一定范围内以剂量依赖方式和时间依赖方式抑制SGC-7901胃癌细胞生长。流式细胞仪检测,SGC-7901细胞经红花多糖处理24 h,其早期凋亡率、细胞坏死或晚期凋亡率显著增加,呈现明显的剂量依赖性。Real-time PCR和Western Blot检测发现,SPS处理的细胞Akt基因及蛋白表达量明显下降。结论 红花多糖对人胃癌SGC-7901细胞体外增殖具有明显的抑制作用,该抑制作用具有一定的时间依赖性和剂量依赖性;红花多糖能够下调Akt mRNA表达,降低Akt和p-Akt蛋白的表达量,抑制Akt通路发挥抗肿瘤作用。     

ZSTK474, a PI3K inhibitor, suppresses proliferation and sensitizes human pancreatic adenocarcinoma cells to gemcitabine

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is important in cell proliferation and survival, and it is frequently and aberrantly activated in pancreatic adenocarcinoma. Potential anti-tumor effect(s) of ZSTK474, a PI3K/Akt inhibitor, together with a key clinically relevant anti-tumor agent, gemcitabine (GEM), have been reported in a human pancreatic cancer xenograft mouse model. However, the precise molecular mechanism of these anti-tumor effects has not been well elucidated. In this study, we investigated the molecular mechanism of GEM plus ZSTK474 in reducing tumor cell survival in human pancreatic cancer cell lines. Our study showed that ZSTK474 inhibited cell growth by arresting cells at the G1 phase and by inducing apoptosis. ZSTK474 also inhibited the phosphorylation of Akt, GSK3β and BAD. The combination of GEM and ZSTK474 demonstrated synergistic anti-tumor effects on pancreatic cancer cells in both transient (3 days) and long-term (14 days) clonogenic assays. Thus, we elucidated the potential molecular mechanism leading to the enhanced anti-tumor effect when GEM and ZSTK474 are combined in treatment.     

PI3K inhibitors in trastuzumab-resistant HER2-positive breast cancer cells with PI3K pathway alterations

The activation of the PI3K signaling pathway resulting from genetic alterations induces carcinogenesis and resistance to anticancer therapies. Breast cancer is a major malignancy that is associated with dysregulation of the PI3K signaling pathway. PIK3CA mutations and PTEN loss occur in every subtype of breast cancer. PI3K inhibitors are being evaluated in breast cancer after the success of an alpha isoform-specific PI3K inhibitor in estrogen receptor (ER)-positive/HER2-negative metastatic breast cancer. Some preclinical data indicate the potential for PI3K/mTOR targeting in combination with trastuzumab for HER2-positive breast cancer with or without expression of the estrogen receptor. However, the role of this therapy in HER2-positive breast cancer with PIK3CA mutations and/or PTEN loss remains unclear. We examined three HER2-positive, ER-negative breast cancer cell lines to determine the efficacy of a novel alpha isoform-specific PI3K inhibitor in combination with trastuzumab. The results indicated that this combination was effective in PIK3CA-mutant or PTEN-deficient breast cancer cells by inducing apoptosis and inhibiting the expression of downstream proteins. PTEN loss by siRNA modulation in parental HER2-positive cancer cells with PI3K signaling pathway alterations could not confer resistance to alpelisib or GDC-0077 plus trastuzumab. We selected the CK-MB-1 cell line without alterations in the PI3K pathway to demonstrate that PI3K inhibitors plus trastuzumab represented a biomarker-specific treatment. In vivo effects of alpelisib plus trastuzumab were tested and confirmed in a mouse model, showing the combination strategy offered the best opportunity to achieve tumor volume reduction. With known safety profiles, this cytotoxic chemotherapy-free regimen warrants further attention as a biomarker-driven strategy for treating HER2-positive breast cancer.     

OPB-31121, a novel small molecular inhibitor,disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells

We investigated the mechanisms of action and antitumor effects of OPB-31121, a novel STAT3 inhibitor, in gastric cancer cells. OPB-31121 downregulated JAK2 and gp130 expression and inhibited JAK2 phosphorylation which leads to inhibition of STAT3 phosphorylation. OPB-31121 inhibited constitutively activated and IL-6-induced JAK/STAT signaling pathway. OPB-31121 decreased cell proliferation in both gastric cancer cells and in a xenograft model, induced the apoptosis of gastric cancer cells, inhibited the expression of antiapoptotic proteins, and showed synergism with 5-fluorouracil and cisplatin. Taken together, our study suggests that STAT3 inhibition with OPB-31121 can be tested in patients with gastric cancer.     

KRAS, BRAF and PIK3CA mutations in human colorectal cancer: relationship with metastatic colorectal cancer

Many abnormal gene expressions and dysregulated signaling pathways have been found in human colorectal cancer. Activating mutations of the KRAS, BRAF or PIK3CA oncogenes are frequently found in colorectal cancer. The aim of the study was to investigate the molecular occurrence of KRAS, BRAF and PIK3CA mutations in the colorectal tumorigenesis and to study the association of these events with clinicopathological parameters. In our study, DNA was extracted from 200 cases of human colorectal cancer tissue samples. KRAS, BRAF and PIK3CA mutation analysis was performed by PCR and pyrosequencing. Using statistical methods, we analyzed the relationships between the gene mutations and clinicopathological parameters. KRAS point mutations were detected in 63/200 patients (31.5%), with codon 12 mutations in 52/200 patients (26%), codon 13 mutations in 10/200 patients (5%) and codon 12.13 bi-mutations in 1/200 patients (0.5%). The V600E mutations of BRAF were detected in 14/200 patients (7%). PIK3CA point mutations (exon 9, exon 20) were detected in 25/200 (12.5%) patients, exon 9 mutatons in 12/200 patients (6%) and exon 20 mutations in 13/200 (6.5%). Our study suggested that both KRAS and BRAF mutations are exclusive, but KRAS and PIK3CA mutations are coexistent. The mutational status of BRAF did not correlate with Dukes' staging, histological type, age and gender. However, strong associations were found between KRAS, PIK3CA mutations and Dukes' staging (staging D, 12/25, 48%). Notably, our data indicated that colorectal cancers with KRAS and PIK3CA bi-mutations are more likely to develop into liver metastasis.     

Gastrin acting on the cholecystokinin2 receptor induces cyclooxygenase-2 expression through JAK2/STAT3/PI3K/Akt pathway in human gastric cancer cells

Gastrin, cholecystokinin2 receptor (CCK2R), and cyclooxygenase-2 (COX-2) have been implicated in the carcinogenesis and progression of gastric cancer. Our study demonstrated that antagonist or siRNA against CCK2R blocked amidated gastrin (G17)-induced activation of STAT3 and Akt in gastric cancer cell lines. G17-increased COX-2 expression and cell proliferation were effectively blocked by CCK2R antagonist and inhibitors of JAK2 and PI3K. In addition, knockdown of STAT3 expression significantly attenuated G17-induced PI3K/Akt activation, COX-2 expression, and cell proliferation. These results suggest that CCK2R-mediated COX-2 up-regulation via JAK2/STAT3/PI3K/Akt pathway is involved in the proliferative effect of G17 on human gastric cancer cells.     

阻断PI3K/AKT通路对乳腺癌细胞放射敏感性影响的研究

目的 研究抑制磷脂酰肌醇3激酶和（或）蛋白激酶B（P13K／AKT）生存传导路径是否改变乳腺癌细胞的放射敏感性。方法 用乳腺癌细胞细胞株MCF7为实验对象，分别接受单纯放射、Ly294002（P13K抑制剂）和二者结合处理。通过Western印记法证实Ly294002可下调AKT活性。采用成克隆法定量分析细胞增殖性死亡。通过半胱天冬酶-3（easpase-3）活性评估细胞凋亡。结果 单纯Ly294002（5μmol／L）可抑制AKT的磷酸化，而单纯放射对AKT的活性无明显影响，二者结合可提高对AKT活性的抑制作用。Ly294002（5μmol／L）在放射前与细胞作用1h及放射后作用10d均可提高MCF7细胞对放射的敏感性。Ly294002结合放射可增加MCF7细胞增殖性死亡，SF4值的放射增敏比为1．25，D0值的放射增敏比为1．42。Ly294002可增加MCF7细胞放射后诱导的细胞凋亡。结论 抑制P13K通过降低AKT活性，增加MCF7细胞对放射的敏感性，为筛选放射敏感剂进行临床实验提供了依据。     

Invention of a novel photodynamic therapy for tumors using a photosensitizing PI3K inhibitor

XL147 (SAR245408, pilaralisib), an ATP‐competitive pan‐class I phosphoinositide 3‐kinase (PI3K) inhibitor, is a promising new anticancer drug. We examined the effect of the PI3K inhibitor on PC3 prostate cancer cells under a fluorescence microscope and found that XL147‐treated cancer cells are rapidly injured by blue wavelength (430 nm) light irradiation. During the irradiation, the cancer cells treated with 0.2–2 μM XL147 showed cell surface blebbing and cytoplasmic vacuolation and died within 15 min. The extent of cell injury/death was dependent on the dose of XL147 and the light power of the irradiation. These findings suggest that XL147 might act as a photosensitizing reagent in photodynamic therapy (PDT) for cancer. Moreover, the cytotoxic effect of photosensitized XL147 was reduced by pretreatment with other ATP‐competitive PI3K inhibitors such as LY294002, suggesting that the cytotoxic effect of photosensitized XL147 is facilitated by binding to PI3K in cells. In a single‐cell illumination analysis using a fluorescent probe to identify reactive oxygen species (ROS), significantly increased ROS production was observed in the XL147‐treated cells when the cell was illuminated with blue light. Taken together, it is conceivable that XL147, which is preferentially accumulated in cancer cells, could be photosensitized by blue light to produce ROS to kill cancer cells. This study will open up new possibilities for PDT using anticancer drugs.