The phosphatidyl inositol 3-kinase/AKT signaling pathway in breast cancer

The phosphatidyl inositol 3-kinase (PI3K)/Akt pathway mediates the effects of a variety of extracellular signals in a number of cellular processes including cell growth, proliferation, and survival. The alteration of integrants of this pathway through mutation of its coding genes increases the activation status of the signaling and can thus lead to cellular transformation. The frequent dysregulation of the PI3K/Akt pathway in breast cancer (BC) and the mediation of this pathway in different processes characteristically implicated in tumorigenesis have attracted the interest of this pathway in BC; however, a more comprehensive understanding of the signaling intricacies is necessary to develop clinical applications of the modulation of this pathway in this pathology. We review a series of experiments examining the contribution of alteration of integrants of this signaling network to human BC and we make an update of the information about the effect of the modulation of this pathway in this cancer.     

磷脂酰肌醇3-激酶在人乳腺癌中的活化

背景与目的：磷脂酰肌醇3-激酶（phosphatidylinsitol 3-kinase,P13-K)可被多种有丝分裂信号刺激活化,它参与有关细胞生长信号的调控,但其在人类肿瘤中的作用尚不清楚。本研究旨在探讨人乳腺癌中P13-K的蛋白表达、mRNA表达和激酶活性变化情况。方法：应用Western blot、免疫沉淀、激酶活性测定和RT-PCR方法检测了37例人乳腺癌及其周围非癌组织中P13-KP85亚单位的表达及P13-K活性变化。结果：与周围非癌组织相比,37例人乳腺癌中,32例（86.49%)P13-Kp85亚单位蛋白表达显著增加;35例（94.59%)P13-Kp85亚单位mRNA水平明显增高;25例（67.57%)p13-k激酶活性亦显著增高。结论：人乳腺癌中P13-K被活化,提示P13-K参与乳腺癌细胞的信号转导;它有可能成为人乳腺癌治疗的新靶点。     

Phosphatidylinositol-3'-kinase/AKT signaling is essential in synovial sarcoma

Synovial sarcomas account for 5-10% of all malignant soft tissue tumors. They have been shown to express different membranous growth factor receptors, many of them signaling via intracellular kinase cascades. In our study, the functional role of PI3K/AKT signals in synovial sarcoma is analyzed with regard to tumor biology and therapeutic applicability. Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT, its targets p-(Ser9)-GSK-3β and p-(Ser2448)-mTOR and the cell cycle regulators Cyclin D1 and p27(KIP1) were performed in 36 synovial sarcomas. The PIK3CA gene was screened for mutations. In vitro, four synovial sarcoma cell lines were treated with the PI3K inhibitor LY294002. Phosphorylation of AKT, GSK-3β and mTOR was assessed, and cellular proliferation and apoptosis were analyzed to functionally characterize the effects of PI3K inhibition. Finally, coincubations of LY294002 with cytotoxic drugs were performed. Most tumors showed significant expression levels of p-AKT, p-GSK-3β and p-mTOR, indicating activation of the PI3K/AKT signaling cascade in synovial sarcomas; Cyclin D1 and p27(KIP1) were differentially expressed. Mutations in the PIK3CA gene could be excluded. In vitro, PI3K inhibition diminished synovial sarcoma cell growth accompanied by reduced phosphorylation of AKT, GSK-3β and mTOR. Mechanistically, PI3K pathway inhibition lead to enhanced apoptosis and decreased cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of synovial sarcoma cell lines with LY294002 and cytotoxic drugs resulted in additive effects. In summary, PI3K signaling plays an essential role in growth control of synovial sarcomas and might be successfully targeted in multimodal therapeutic strategies.     

p53 mutations occur in aggressive breast cancer.

Using a polymerase chain reaction-single strand conformation polymorphism approach we analyzed 96 human primary breast tumors for the presence of mutations in exons 2, 5, 6, 7, 8, and 9 of the p53 gene. These exons have been shown to comprise highly conserved sequences and the portion including exons 5 through 9 is believed to be the target for over 90% of the acquired mutations in human cancer. Eighteen tumors of the 96 (18.7%) tested showed reproducibly a variant band indicative of a mutation. Most (15 tumors) of the mutations were single nucleotide substitutions and G:C to A:T transitions were prevalent (6 tumors), G:C to T:A transversions came next (4 tumors), and guanines were always on the nontranscribed strand. Concomitant loss of the wild type allele and mutation of the other copy was observed in only 3 of 18 mutated cases; this is consistent with the heterogeneous cellular composition of breast tumors. Furthermore p53 mutations were correlated to estrogen and/or progesterone receptor negative tumors, thus indicating their relationships to aggressive breast cancer. No association could be observed with DNA amplification events in these tumors.     

RNAi下调AKT1、PI3K P85表达抑制乳腺癌MCF-7细胞的增殖

目的：探讨RNAi（RNA interference）技术抑制乳腺癌MCF7细胞中AKT1和PI3K P85亚基的表达对MCF7细胞增殖和侵袭等的影响。方法：将包含AKT1、PI3K P85两种siRNA开放阅读框的短发夹RNA（shRNA）重组腺病毒质粒表达载体rAd5siAKT1siPI3K转染至乳腺癌MCF7细胞。应用realtime PCR和Western blotting检测转染后目的基因mRNA和蛋白的表达水平,并用Western blotting检测目的基因被沉默后PCNA、cyclin D1和P53的表达情况。应用MTT法、流式细胞术、2D和3D Matrigel实验检测MCF7细胞转染前后的细胞增殖周期和侵袭能力。结果：重组腺病毒质粒表达载体rAd5siAKT1siPI3K介导的靶向〖STBX〗AKT1, PI3K P85 shRNA可以有效抑制目的基因AKT1和PI3 Kp85的mRNA和蛋白表达;下游相关因子PCNA、cyclin D1的表达亦下调,P53表达则上调。MTT法结果显示rAd5siAKT1siPI3K组细胞生长抑制率>50%,与未转染组和rAd5siCtrl转染组比较,出现明显的G1/G0细胞周期阻滞;2D和3D Matrigel实验显示,未转染组和rAd5siCtrl转染组细胞呈正常形态,而rAd5siAKT1siPI3K 转染组细胞贴壁生长能力明显减低,细胞团块明显缩小。结论：靶向AKT1、PI3K P85亚基的shRNA技术可以抑制MCF7细胞中AKT1、PI3K P85亚基的表达,抑制MCF7细胞的体外增殖。     

Spectrum of PIK3CA/AKT mutations across molecular subtypes of triple-negative breast cancer

Breast Cancer Research and Treatment - The heterogeneity of triple-negative breast cancer (TNBC) confers variable response to chemotherapy that results in poor outcome and relapse. Due to lack of...     

An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.     

Phosphatidylinositol-3-OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase I inhibitor-induced apoptosis

Geranylgeranyltransferase I inhibitors (GGTIs) represent a new class of anticancer drugs. However, the mechanism by which GGTIs inhibit tumor cell growth is still unclear. Here, we demonstrate that GGTI-298 and GGTI-2166 induce apoptosis in both cisplatin-sensitive and -resistant human ovarian epithelial cancer cells by inhibition of PI3K/AKT and survivin pathways. Following GGTI-298 or GGTI-2166 treatment, kinase levels of PI3K and AKT were decreased and survivin expression was significantly reduced. Ectopic expression of constitutively active AKT2 and/or survivin significantly rescue human cancer cells from GGTI-298-induced apoptosis. Previous studies have shown that Akt mediates growth factor-induced survivin, whereas p53 inhibits survivin expression. However, constitutively active AKT2 failed to rescue the GGTIs downregulation of survivin. Further, GGTIs suppress survivin expression and induce programmed cell death in both wild-type p53 and p53-deficient ovarian cancer cell lines. These data indicate that GGTI-298 and GGTI-2166 induce apoptosis by targeting PI3K/AKT and survivin parallel pathways independent of p53. Owing to the fact that upregulation of Akt and survivin as well as inactivation of p53 are frequently associated with chemoresistance, GGTIs could be valuable agents to overcome antitumor drug resistance.     

Germline BRCA1 and HMLH1 mutations in a family with male and female breast carcinoma

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease, predisposing to the development of colorectal cancer and other tumor types such as endometrial, small bowel, stomach, ovary and urinary tract carcinoma, while most investigators find no association between HNPCC and cancer of the breast. We have identified hMLH1 mutations in 2 Amsterdam-criteria HNPCC families where both male and female gene carriers were affected with breast cancer. To investigate whether these breast cancers were caused by other genetic factors, we analyzed the 2 breast cancer susceptibility genes BRCA1 and BRCA2. In one family we did not find any mutation in the breast cancer genes, while in the other, a BRCA1 mutation segregated in the breast cancer cases. Hereditary breast cancer, and in particular BRCA1 tumors, display discrete histo-pathological and immunohistological characteristics. The tumor from a woman with both hMLH1 mutations and a BRCA1 mutation exhibited typical BRCA1 histology, e.g., grade 3 invasive ductal carcinoma with dense lymphocytic infiltration, and immunohistology, estrogen receptor (ER) negative, progesterone receptor (PgR) negative, strongly p53 positive, c-erbB-2 negative and highly Ki67 positive (>50% stained cells). The histology of the breast tumor from the man with both one hMLH1 mutation and a BRCA1 mutation was a grade 2 invasive ductal carcinoma without any special BRCA1 features. Immunohistology was also different. This might merely reflect a true difference in male breast tumor progression vs. female. We cannot exclude that the combined effect of BRCA1 and hMLH1 dysfunction has a bearing on tumor progression.     

Increased frequency of ATM mutations in breast carcinoma patients with early onset disease and positive family history

BACKGROUND: An increased incidence of breast carcinoma has been reported among relatives of individuals who are affected with the rare recessive disorder, ataxia-telangiectasia (A-T), and who are heterozygous for mutations in the ataxia-telangiectasia mutated (ATM) gene. However, most studies of breast carcinoma cases from the general population have failed to find a higher incidence of ATM mutations in cases when compared with controls. METHODS: Genomic DNA samples from 258 individuals were screened for mutations of all types in each of the 62 coding exons of the ATM gene; 142 of these were from breast carcinoma cases with a first-degree family history or early age at diagnosis, 35 were from cases selected for the presence of either known disease-related mutations (n = 25) or missense alterations of unknown consequences (n = 10) in BRCA1 or BRCA2, and 81 were from matched controls. RESULTS: A total of 12 individuals with ATM mutations were identified, 11 among 142 breast carcinoma cases (7.7%; 95% CI, 3.9-13.4%) and 1 among 81 controls (1.2%; 95% CI, 0.0-6.7%) (P = 0.06). All mutations detected were of the missense type; none were predicted to truncate the ATM protein. Among cases, mutations were found exclusively in patients with a family history of breast carcinoma (12.1%; 95% CI, 6.2-20.6%) (P = 0.02). Similar frequencies of ATM mutations were found in 35 additional cases selected for the presence of BRCA1 or BRCA2 mutations when compared with cases overall. CONCLUSIONS: ATM mutations, specifically missense mutations, are more common in breast carcinoma cases selected for first-degree family history and early age at diagnosis.     

Mutational analysis of AKT1, AKT2 and AKT3 genes in common human carcinomas

OBJECTIVE: Mounting evidence indicates that alterations in AKT proteins play an important role in the pathogenesis of cancer. The objective of this study was to see whether common human carcinomas harbor AKT mutations that might contribute to the development of cancer. METHODS: We performed mutational analysis of the kinase domains of AKT1-AKT3 by a single-strand conformation polymorphism assay in 294 carcinoma tissues from the stomach, lung, colon and breast. RESULTS: Overall, we detected three somatic mutations in AKT2, but no mutations in AKT1 or AKT3 in the 294 cancer tissues. The AKT2 mutations were detected in 1 of 51 gastric carcinomas (2.0%) and 2 of 79 lung carcinomas (2.5%). AKT2 mutations consisted of one missense mutation and 2 splice site mutations in the intron. We simultaneously analyzed somatic mutations in EGFR, ERBB2, K-RAS, PIK3CA and BRAF genes in the 3 samples with the AKT2 mutations, and found a lung adenocarcinoma with the AKT2 missense mutation harbored an EGFR mutation. CONCLUSION: This study demonstrated that somatic mutations in the kinase domain of AKT2 occur in a small fraction of common human cancers, and suggested that alterations in the AKT2-mediated signaling pathway by AKT2 mutation could contribute to the development of some cases of human cancers.     

三阴性乳腺癌中PI3K/AKT/mTOR信号通路的突变及相关靶向治疗的研究进展

三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型,PI3K/AKT/mTOR信号通路失调是TNBC最常见的致癌突变之一,靶向PI3K/AKT/mTOR信号通路是治疗TNBC的重要方向。本文着重介绍了PI3K/AKT/mTOR信号通路的机制,TNBC中出现的PIK3CA、AKT1或mTOR的突变,以及失活张力PTEN、PIK3R1或INPP4B的突变或丢失,也展现了布帕尼西、帕他色替、依维莫司等PI3K/AKT/mTOR信号通路靶向药物在治疗TNBC中单独、联合应用和与化疗或免疫疗法联用的疗效,同时论述了目前正在进行的各类临床试验及其未来的前景。