Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.     

Significance of PIK3CA Mutations in Patients with Early Breast Cancer Treated with Adjuvant Chemotherapy: A Hellenic Cooperative Oncology Group (HeCOG) Study

Background

The PI3K-AKT pathway is frequently activated in breast cancer. PIK3CA mutations are most frequently found in the helical (exon 9) and kinase (exon 20) domains of this protein. The aim of the present study was to examine the role of different types of PIK3CA mutations in combination with molecular biomarkers related to PI3K-AKT signaling in patients with early breast cancer.

Methods

Tumor tissue samples from 1008 early breast cancer patients treated with adjuvant chemotherapy in two similar randomized trials of HeCOG were examined. Tumors were subtyped with immunohistochemistry (IHC) and FISH for ER, PgR, Ki67, HER2 and androgen receptor (AR). PIK3CA mutations were analyzed by Sanger sequencing (exon 20) and qPCR (exon 9) (Sanger/qPCR mutations). In 610 cases, next generation sequencing (NGS) PIK3CA mutation data were also available. PIK3CA mutations and PTEN protein expression (IHC) were analyzed in luminal tumors (ER and/or PgR positive), molecular apocrine carcinomas (MAC; ER/PgR negative / AR positive) and hormone receptor (ER/PgR/AR) negative tumors.

Results

PIK3CA mutations were detected in 235/1008 tumors (23%) with Sanger/qPCR and in 149/610 tumors (24%) with NGS. Concordance between the two methods was good with a Kappa coefficient of 0.76 (95% CI 0.69–0.82). Lobular histology, low tumor grade and luminal A tumors were associated with helical domain mutations (PIK3CAhel), while luminal B with kinase domain mutations (PIK3CAkin). The overall incidence of PIK3CA mutations was higher in luminal as compared to MAC and hormone receptor negative tumors (p = 0.004). Disease-free and overall survival did not significantly differ with respect to PIK3CA mutation presence and type. However, a statistically significant interaction between PIK3CA mutation status and PTEN low protein expression with regard to prognosis was identified.

Conclusions

The present study did not show any prognostic significance of specific PIK3CA mutations in a large group of predominantly lymph-node positive breast cancer women treated with adjuvant chemotherapy. Further analyses in larger cohorts are warranted to investigate possible differential effect of distinct PIK3CA mutations in small subgroups of patients.     

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background

Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and Methods

PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results

PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions

Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.     

PIK3CA mutations in human solid tumors: Role in sensitivity to various therapeutic approaches

Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival and motility. The PI3K pathway is considered to play an important role in tumorigenesis. Activating mutations of the p110α subunit of PI3K (PIK3CA) have been identified in a broad spectrum of tumors. Analyses of PIK3CA mutations reveals that they increase the PI3K signal, stimulate downstream Akt signaling, promote growth factor-independent growth and increase cell invasion and metastasis. In this review, we analyze the contribution of the PIK3CA mutations in cancer, and their possible implications for diagnosis and therapy.     

Network Analysis Reveals A Signaling Regulatory Loop in PIK3CA-mutated Breast Cancer Predicting Survival Outcome

Mutated genes are rarely common even in the same pathological type between cancer patients and as such, it has been very challenging to interpret genome sequencing data and difficult to predict clinical outcomes. PIK3 CA is one of a few genes whose mutations are relatively popular in tumors. For example, more than 46.6% of luminal-A breast cancer samples have PIK3 CA mutated, whereas only 35.5% of all breast cancer samples contain PIK3 CA mutations. To understand the function of PIK3 CA mutations in luminal A breast cancer, we applied our recentlyproposed Cancer Hallmark Network Framework to investigate the network motifs in the PIK3CA-mutated luminal A tumors. We found that more than 70% of the PIK3CA-mutated luminal A tumors contain a positive regulatory loop where a master regulator(PDGF-D), a second regulator(FLT1) and an output node(SHC1) work together. Importantly, we found the luminal A breast cancer patients harboring the PIK3 CA mutation and this positive regulatory loop in their tumors have significantly longer survival than those harboring PIK3 CA mutation only in their tumors. These findings suggest that the underlying molecular mechanism of PIK3 CA mutations in luminal A patients can participate in a positive regulatory loop, and furthermore the positive regulatory loop(PDGF-D/FLT1/SHC1) has a predictive power for the survival of the PIK3 CAmutated luminal A patients.     

PIK3CA基因突变的MassARRAY分子量阵列分析

目的：利用MassARRAY分子量阵列分析系统检测胃癌组织PIK3CA基因突变。方法：从胃癌石蜡包埋组织中提取基因组,PCR反应扩增目的基因片段,MassARRAY分子量阵列分析系统检测PIK3CA基因突变;焦磷酸测序验证检测结果。结果：中国西部地区144例胃癌组织样本中PIK3CA_E542K（1624G〉A）突变携带率为77．6％,PIK3C_LIE545K（1633G〉A）突变携带率为84％。MassARRAY分子量阵列分析系统检测结果与焦磷酸测序结果达到100％吻合。结论：建立了MassARRAY分子量阵列分析系统检测基因突变的方法,初步建立了中国西北地区汉族人群胃癌组织PIK3CA基因PIK3CA_E542K（1624G〉A）PIK3CA_E545K（1633G〉A）位点突变频数。     

Analysis of Driver Mutations in Female Non-Smoker Asian Patients with Pulmonary Adenocarcinoma

Previous studies have revealed that EGFR mutation and/or EML4?CALK gene fusion rate was higher in the non-smoker Asian females with pulmonary adenocarcinoma. The aim of this study is to determine the distribution of known oncogenic driver mutations in the female non-smoker Asian patients with pulmonary adenocarcinoma. 104 consecutively resected lung adenocarcinomas from 396 non-smoker females (less than 100 cigarettes in a lifetime) at a single institution (Tongji University, Shanghai, China) were analyzed for mutations in EGFR, EML4?ALK, KRAS, HER2, BRAF, and PIK3CA. 73 (70.2?%) tumors harbored EGFR mutations; among these, 28 were deletions in exon 19, 44 were L858R missense changes, and eight were T790M mutations. 10 (9.6?%) harbored EML4?ALK fusions, two harbored KRAS mutations, two harbored BRAF mutations, and two harbored PI3K mutations. A majority of the mutations were mutually exclusive, except two with EGFR mutation and BRAF mutation, one with EML4?ALK fusions and PI3K mutation. Thus, 82.7?% (86 of 104; 95?% CI, 75.4?C90.0?%) of lung adenocarcinomas from non-smoker females were found to harbor the well-known oncogenic mutations in five genes. Lung cancer in non-smoking Asian females is a distinct entity, with majority of this subgroup being developed by the oncogenic mutations. The prospective mutation examination in this population will be helpful for devising a targeted therapy for a majority of the patients.     

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.     

Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110α catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110α, p110β, and p110δ, each coexpressed with the regulatory subunit p85α or splice variant p55α. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.     

Oncogenic Mutations of PIK3CA in Human Cancers

Phosphatidylinositol 3-kinases (PI3Ks) are important regulators of signaling pathways. To determine whether PI3Ks are genetically altered in human cancers, we recently analyzed the sequences of the PI3K gene family and discovered that one member, the PIK3CA gene encoding the p110? catalytic subunit, was frequently mutated in cancers of the colon, breast, brain and lung. The majority of mutations clustered near two positions within the PI3K helical or catalytic domains and at least one hotspot mutation appeared to increase kinase activity. PIK3CA represents one of the most highly mutated oncogenes identified in human cancers and may be a useful diagnostic and therapeutic target.     

Activation of the PI3K/AKT pathway in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients.     

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.     

An Integrative Analysis of PIK3CA Mutation,PTEN, and INPP4B Expression in Terms of Trastuzumab Efficacy in HER2-Positive Breast Cancer

The phosphoinositide-3-kinase (PI3K) pathway is commonly deregulated in breast cancer through several mechanisms, including PIK3CA mutation and loss of phosphatase and tensin homolog (PTEN) and inositol polyphosphate 4-phosphatase-II (INPP4B). We aimed to evaluate the predictive relevance of these biomarkers to trastuzumab efficacy in HER2-positive disease. We evaluated the effect of trastuzumab in 43 breast cancer patients with HER2-overexpression who received neoadjuvant treatment. PIK3CA mutation was examined by direct sequencing and digital PCR assay, and PIK3CA copy number was assessed by digital PCR assay of pretreatment tissues. PTEN, pAkt, and INPP4B were assessed by immunohistochemistry. Direct sequencing detected mutant DNA in 21% of all patients, but the incidence increased to 49% using digital PCR. The pathological complete response (pCR) rate in patients with PIK3CA mutations was 29% compared with 67% for those without PIK3CA mutations (P = 0.093), when the mutation was defined as positive if the mutant proportion was more than 10% of total genetic content by digital PCR. Low PTEN expression was associated with less pCR compared to high expression (33% versus 72%, P = 0.034). There were no significant associations of PIK3CA copy number, pAKt, or INPP4B with trastuzumab efficacy. In multivariate analysis, activation of the PI3K pathway due to either PIK3CA mutation or low PTEN were related to poorer response to trastuzumab (OR of predictive pCR was 0.11, 95%CI; 0.03–0.48). In conclusion, activating the PI3K pathway is associated with low pCR to trastuzumab-based treatment in HER2-positive breast cancer. Combined analysis of PIK3CA mutation and PTEN expression may serve as critical indicators to identify patients unlikely to respond to trastuzumab.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

Novel Somatic Mutations to PI3K Pathway Genes in Metastatic Melanoma

Background

BRAF^V600 inhibitors have offered a new gateway for better treatment of metastatic melanoma. However, the overall efficacy of BRAF^V600 inhibitors has been lower than expected in clinical trials, and many patients have shown resistance to the drug’s effect. We hypothesized that somatic mutations in the Phosphoinositide 3-Kinase (PI3K) pathway, which promotes proliferation and survival, may coincide with BRAF^V600 mutations and contribute to chemotherapeutic resistance.

Methods

We performed a somatic mutation profiling study using the 454 FLX pyrosequencing platform in order to identify candidate cancer genes within the MAPK and PI3K pathways of melanoma patients. Somatic mutations of theses candidate cancer genes were then confirmed using Sanger sequencing.

Results

As expected, BRAF^V600 mutations were seen in 51% of the melanomas, whereas NRAS mutations were seen in 19% of the melanomas. However, PI3K pathway mutations, though more heterogeneous, were present in 41% of the melanoma, with PTEN being the highest mutated PI3K gene in melanomas (22%). Interestingly, several novel PI3K pathway mutations were discovered in MTOR, IRS4, PIK3R1, PIK3R4, PIK3R5, and NFKB1. PI3K pathway mutations co-occurred with BRAF^V600 mutations in 17% of the tumors and co-occurred with 9% of NRAS mutant tumors, implying cooperativity between these pathways in terms of melanoma progression.

Conclusions

These novel PI3K pathway somatic mutations could provide alternative survival and proliferative pathways for metastatic melanoma cells. They therefore may be potential chemotherapeutic targets for melanoma patients who exhibit resistance to BRAF^V600 inhibitors.     

Mutations in TP53 and PIK3CA genes in hepatocellular carcinoma patients are associated with chronic Schistosomiasis

The purpose of this study was to evaluate the genetic variation of the PIK3CA gene and the histopathological changes in liver tissue of patients with chronic Schistosomiasis to predict hepatocellular carcinoma. In this retrospective, the study samples were taken from 20 patients, divided into chronic schistosomiasis infected group of people (S) and chronic schistosomiasis uninfected group of people (C). The liver tissue biopsy samples for histological examinations were obtained only from chronic Schistosomiasis patients (n = 9). The blood samples were obtained from groups S and C for the mutational analysis of the PIK3CA and TP53 genes. The results suggest that the patients diagnosed with chronic Schistosomiasis were 9 (55%), and healthy patients without Schistosomiasis were 11 (45%). Histological results found that proliferation of fibrosis was observed in the hepatocytes of schistosomiasis patients. A total of 8 mutations (5 male, 3 female) were detected in PIK3CA and TP53 genes. Including 1634 A > G substitution mutations in PIK3CA, which was the only mutation found in males and females among the 8 mutations, accounting 22.22%. PIK3CA gene mutations were found more predominant in male groups as compared to other TP53 gene mutations. In conclusion, this study found that patients with chronic Schistosomiasis are at risk of PIK3CA gene mutations, eventually leading to hepatocytes fibrosis and liver cancer.     

Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor,ARQ 092

As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.