H1047R Mutation Research Articles

Abstract 5249: Butterfly effect in cancer: How a single base mutation of PIK3CA remodels multiple pathways

Abstract PI 3-kinase is a frequently mutated oncogene. Along with the phosphatase PTEN, numerous receptor tyrosine kinases, and Akt, the PI 3-kinase pathway is among the most frequently activated in cancer. The H1047R mutation of PIK3CA is a representative gain-of-function mutation in the PI 3-kinase pathway and is transforming in both chicken fibroblasts and mouse embryonic fibroblasts. Recently, a knock-in of this mutation has been made in the normal human mammary epithelial cell line, MCF-10A. Here, H1047R confers growth factor independence, but does enable the knock-in cells to grow in soft agar or to form xenografts in mice. We have investigated this knock-in cell line and the parental MCF-10A by both deep sequencing and proteomics to determine the transcriptional and translational changes induced by PIK3CA-H1047R. We can confirm that the only coding mutation introduced into the MCF-10A cell line is PIK3CA-H1047R. Surprisingly, this single base mutation induces changes in the expression of thousands of transcripts and proteins. Despite rapid proliferation and growth factor independence, H1047R also induces a decrease in cell cycle genes including Cyclin A2 and Cyclin D2. The decrease in these genes may be the cause of an observed decrease in S and G2 phase cells as compared with the parental MCF-10A cell line. The parental cells overexpress MYC, however this overexpression is lost upon H1047R expression. Inhibition of PI 3-kinase with small molecule compounds that are selective for p110α or affect all isoforms of p110 as well as inhibition of mTOR do not restore the expression of c-MYC. This suggests that long-term activation of the PI 3-kinase pathway can result in changes to the gene expression landscape which no longer depend upon the PI 3-kinase pathway. Finally, H1047R causes changes in 3D cell culture morphology. Both MCF-10A and the H1047R knock-in form acinar structures with hollow lumen when grown on reduced growth factor Matrigel substrates. However, if these cultures are treated with IGF1, the MCF-10A cells retain their acinar structures while, H1047R form branching tube-like structures which invade into Matrigel. The formation of these structures can be inhibited by the introduction of GDC-0941, a pan-PI3K inhibitor. IGF1 has no effect upon cell proliferation or migration in 2D cell culture systems. In summary, the H1047R mutation of PIK3CA while possessing a single catalytic activity can lead to a diverse range of phenotypes beyond growth factor independence. This work was supported by the National Institutes of Health under award numbers R01 CA078230 and R21 AG039716 (JRH and PKV), P30 NS057096 and P41 RR011823 (YZ, LL and JRY). This is abstract 24060 of The Scripps Research Institute. Citation Format: Jonathan R. Hart, Yaoyang Zhang, Lujian Liao, Lisa Du, John R. Yates, Peter K. Vogt. Butterfly effect in cancer: How a single base mutation of PIK3CA remodels multiple pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2014-5249

Abstract 3925: The PIK3CA H1047R mutation can initiate tumorigenesis in the colon

Abstract Background: Our understanding of how mutations within the intestinal epithelium result in tumor development and progression is continuously evolving. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K resulted in invasive mucinous adenocarcinomas in the proximal colon through a non-canonical mechanism. We sought to determine if the hotspot PIK3CA H1047R mutation, commonly found in human colorectal cancers, can also induce intestinal tumorigenesis. Methods: Female FVB mice carrying a conditional PIK3CA H1047R mutation (PIK3CAH1047R) were crossed to B6 males carrying atransgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3CAH1047R progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon. 22 F1 FC PIK3CAH1047R mice were examined. Necropsy was performed at approximately 200 days of age or once moribund. Results: 50% of FC PIK3CAH1047R mice develop invasive adenocarcinomas within the colon. These cancers are highly mucinous and often penetrate through the serosal layer of the colon. These lesions can be found throughout the colon, but are more common proximally. No tumors were identified within the small intestine. These cancers can have associated invasion into the regional lymphatics and metastases to the mesentery. Interestingly these cancers have a minimal luminal component compared to the amount of submucosal extension. Precursor lesions are able to be identified and are not polypoid. These cancers demonstrate activation of the AKT signaling pathway, but do not demonstrate nuclear localization of β-catenin. Conclusion: This is the first report of the hotspot PIK3CA H1047R mutation leading to intestinal tumorigenesis through a non-canonical mechanism, resulting in flat mucinous invasive adenocarcinomas. Citation Format: Cheri Pasch, Molly Maher, Dustin Deming. The PIK3CAH1047R mutation can initiate tumorigenesis in the colon. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2014-3925

Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer

Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire–mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6–300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (Km =110 µM) compared with the R132H homodimer (Km = 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).

The Role of Targeted Therapy and Oncogene Dependence on the PIK3CA Mutation

Objectives: (1) Determine the role of oncogene dependence on one of the more common and targetable oncogenes in head and neck squamous cell carcinoma (HNSCC) PIK3CA. (2) Evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Methods: A basic science study was performed to test the hypothesis that PI3KCA hot spot mutations confer increased sensitivity or oncogene addiction to PI3K and related inhibitors. The PI3KCA hotspot mutations—E545K and H1047R—were engineered within the HNSCC cell line SCC25. Cell viability was measured using high throughput microscopy to determine the survivability cell lines expressing the hotspot mutations compared with control cell lines when treated with increasing concentrations of newly developed targeted therapies 17-AAG, GDC-0941, and Trametinib. Results: Surprisingly, hotspot E545K and H1047R mutations conferred increased rather than reduced survivability when treated with increased concentrations of the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and Trametinib compared to the SCC25 control cell lines. Conclusions: (1) The PIK3CA mutations within our engineered cell model did not lead to oncogene dependent cell death when treated with targeted therapy including direct inhibition of the Phosphatidylinositol 3-kinase (PI3K) enzyme. (2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified.

Hotspot mutations in common oncogenes are infrequent in nasopharyngeal carcinoma.

Oncogene mutations contribute to carcinogenesis and can provide potential therapeutic targets for clinical anticancer management. However, oncogene mutation patterns in nasopharyngeal carcinoma(NPC) have yet to be fully elucidated. To gain insight into mutation patterns in NPC, a high-throughput OncoCarta panel assay was used to determine 238 hotspot mutations across 19 common oncogenes in 8 NPC cell lines and 160 NPC patient samples from southern China. Statistical analyses were further conducted to identify associations between oncogene mutations and selected clinicopathological characteristics. In total, we identified 24 mutations across 11 oncogenes in 17 (10.6%) NPC patients. Four patients exhibited mutations in at least one oncogene. We also identified a PIK3CA H1047R mutant in 7 NPC cell lines. In addition, oncogene mutations showed no correlation with either risk habits (smoking and drinking) or other clinical characteristics except for TNM stage. KIT mutations were associated with poorer overall and relapse-free survival. Furthermore, KIT mutations together with age and N stage were independent prognostic factors in NPC. Taken together, the present study is the first report on mutations in multiple oncogenes in NPC. We found that hotspot oncogene mutations are infrequent in NPC patients from southern China. The lack of hotspot mutations requires a comprehensive characterization of gene mutations in NPC for developing new therapeutic targets in the future.

RAPID IDH1 GENE MUTATION ANALYSIS FOR INTRAOPERATIVE PATHOLOGICAL DIAGNOSIS

BACKGROUND: (blind field). METHODS: DNA extracted in 15 min from glioma tissue collected during surgery was used to test for R132 point mutations of the IDH1 gene by using real-time PCR/high resolution melting analysis method (LightCycler 480 system). Normally, detecting IDH1 mutations requires about 80 min from the start of the PCR cycle. For rapid diagnosis, however, DNA extension and annealing times in the PCR cycle were reduced by half. Our results were compared with those obtained using the regular method. RESULTS: Regular analysis and rapid diagnosis analysis were used to detect IDH1 mutations in 6 glioma cases (diffuse astrocytoma, 2 cases; oligodendroglioma, 2 cases; anaplastic astrocytoma, 1 case; glioblastoma, 1 case). Both methods produced the same results in all cases. CONCLUSIONS: Direct DNA sequencing and immunostaining can be used to identify IDH1 mutations. However, because analyses by using the former method require several hours, it cannot be used in rapid diagnosis. The latter method could not identify all R132 mutations, because the current commercially available antibodies can only detect R132H mutant proteins. This method can detect IDH1 mutation in even 1% of tumor purity, and determine all IDH1 R132 mutation-positive gliomas during surgery in 50–60 min after the tissue is collected. Further, this method could aid in intraoperative pathological diagnoses to differentiate IDH1 mutation-positive low-malignancy gliomas from gliosis and other non-neoplastic tissues. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Mutant p53 reactivation by small molecules makes its way to the clinic.

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA-1 and its analog APR-246 that affect a wider range of mutant p53 proteins. APR-246 has been tested in a Phase I/II clinical trial with promising results.

Analysis of PIK3CA Mutations in Breast Cancer Subtypes

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) is a central element of a signaling pathway involved in cell proliferation, survival, and growth. Certain mutations in this pathway result in enhanced PI3K signaling, which is associated with oncogenic cellular transformation and cancer. The aims of this study were to characterize different types of PIK3CA mutations in exons 9 and 20 in a series of primary breast carcinomas and to correlate the results with clinicopathologic parameters and survival. We used frozen tissue samples and sequenced exons 9 and 20 for a series of 241 patients with a diagnosis of breast carcinoma. We found that 15.8% of the primary breast carcinomas possessed PIK3CA mutations in either exon 9 or exon 20. The rate of PIK3CA mutations was increased in HR(+)/HER2(-) tumors (18.6%), but this difference did not reach a statistical significance. The lowest rate of mutations was observed in HR(+)/HER2(+) tumors (5.3%). No statistically significant association was found between the presence of PIK3CA mutations and the prognostic/clinical features of breast cancer, including histologic subtype, Her2 status, axillary lymph node involvement, tumor grade, and tumor stage. However, the presence of the H1047R mutation in 10 samples was associated with a statistically significantly worse overall survival. PIK3CA mutation was found to be a frequent genetic change in all breast cancer subtypes but occurred with the highest rate in HR(+)/HER2(-) tumors. Further studies are needed to validate the prognostic impact of different PIK3CA mutations.

Abstract P1-04-04: Towards methodology of detecting PIK3CA mutations on single cell level by massive parallel sequencing

Abstract Background: The oncogene PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase. Gain-of-function mutations occur in 20 – 40% of the diagnosed breast cancer cases and may predict response to PI3K/AKT/mTOR inhibitors. We have developed a methodology to determine the PIK3CA exon 9 and 20 mutational status on single cell level, allowing analysis of low-input patient material such as circulating tumor cells (CTC). Methods: Single and groups of cultured breast cancer cells (BT20, MDA-MB-361 and MCF7) were isolated and recovered from spiked blood samples using the CellSearch and DEPArray platform respectively, and subjected to whole-genome amplification. Additionally, Formalin-fixed, paraffin-embedded (FFPE) tissue sections from 3 breast cancer patients were collected, of which two were mutant (MT) and one carried the wild-type (WT) genotype, as previously determined by external laboratories. Targeted PIK3CA PCR for exon 9 and 20 with unique multiplex identifiers (MIDs) allowed mixing of 32 samples and differentiation in the sequence data. Massive parallel sequencing was carried out using Titanium Amplicon chemistry (Lib-A) on a 454 GS Junior and data were analyzed using the amplicon variant analyzer (AVA) software. Results: A throughput of 120.464 high-quality reads with a mean coverage depth of 987 reads (range 288 – 5979) allowed the reliable assessment of variants, which were concordant with NCBI SNP/COSMIC databases and external laboratories. In BT20, the variants and frequencies were the missense P539R (48,83%) and H1047R (45,36%) mutations. Missense E545K mutation was detected in MDA-MB-361 (33,33%) and MCF7 (70,83%). Above mentioned mutations were detected in 73,3% and 100% of analyzed single and groups of tumor cells, respectively. Inability to determine the mutational status in some single cells was due to loss of amplicon during purification. In the MT FFPE tissue sections, two variants were detected and were concordant with previously obtained results. The variants and frequencies detected were E542K (14,12%) and E545K (21,38%), and correlated well with the tumor cell content of the actual sample (40% and 50%, respectively). Additionally, variant analysis showed 2 different SNPs in exon 20, which are classified as the synonymous mutations T1025T and A995A. In the WT sample no PIK3CA mutations were detected, suggesting a 100% specificity on the obtained 454 data. Conclusions: Our study delivers a semiautomated workflow using next-generation sequencing for the assessment of PIK3CA gain-of-function mutations on FFPE tissue sections and on single DEPArray purified cells from CellSearch pre-enriched whole blood samples. We are now implementing this workflow in the analysis of circulating tumor cells from patients with metastatic breast cancer. At the time of submitting, a cohort of 40 patients with luminal breast cancer was selected to assess the PIK3CA mutational status in the primary tumor, CTC and (if present) metastatic compartment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-04.

Loss of PTEN Expression Is Associated with Poor Prognosis in Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas

Previously, we reported PIK3CA gene mutations in high-grade intraductal papillary mucinous neoplasms (IPMN). However, the contribution of phosphatidylinositol-3 kinase pathway (PI3K) dysregulation to pancreatic carcinogenesis is not fully understood and its prognostic value unknown. We investigated the dysregulation of the PI3K signaling pathway in IPMN and its clinical implication. Thirty-six IPMN specimens were examined by novel mutant-enriched sequencing methods for hot-spot mutations in the PIK3CA and AKT1 genes. PIK3CA and AKT1 gene amplifications and loss of heterozygosity at the PTEN locus were also evaluated. In addition, the expression levels of PDPK1/PDK1, PTEN, and Ki67 were analyzed by immunohistochemistry. Three cases carrying the E17K mutation in the AKT1 gene and one case harboring the H1047R mutation in the PIK3CA gene were detected among the 36 cases. PDK1 was significantly overexpressed in the high-grade IPMN versus low-grade IPMN (P = 0.034) and in pancreatic and intestinal-type of IPMN versus gastric-type of IPMN (P = 0.020). Loss of PTEN expression was strongly associated with presence of invasive carcinoma and poor survival in these IPMN patients (P = 0.014). This is the first report of AKT1 mutations in IPMN. Our data indicate that oncogenic activation of the PI3K pathway can contribute to the progression of IPMN, in particular loss of PTEN expression. This finding suggests the potential employment of PI3K pathway-targeted therapies for IPMN patients. The incorporation of PTEN expression status in making surgical decisions may also benefit IPMN patients and should warrant further investigation.

Analysis of mutations in the PIK3CA and FGFR3 genes in verrucous epidermal nevus

Verrucous epidermal nevi are congenital hamartomas composed of keratinocytes and may occur alone or in association with developmental abnormalities. A close relationship between variations in the PIK3CA and FGFR3 genes and the appearance of nevi has been recently reported. Based on that, we performed molecular assays for the identification of E542K, E545G/K and H1047R mutations in the PIK3CA gene and of the R248C mutation in the FGFR3 gene. Interestingly, during the amplification process, we did not observe the PCR product of exon 9 of the PIK3CA gene, a region comprising amino acids 542-545. This strongly suggests the occurrence of a microdeletion of that region and indicates a possible allelic variant, which has not yet being described in the literature.

Frequent PIK3CA Mutations in Radial Scars

Radial scars are breast lesions of uncertain pathogenesis that are associated with a 2-fold increased risk of breast cancer compared with that in controls. Activating point mutations in PIK3CA are found in 25% to 30% of invasive breast cancers; however, they have not previously been investigated in radial scars. We sought to evaluate radial scars for known activating point mutations commonly seen in invasive breast cancer. Sixteen surgical cases containing 22 radial scars were identified from pathology archives. Lesional tissue was macrodissected from unstained paraffin sections; genomic DNA was then extracted and screened for a panel of known hotspot mutations using polymerase chain reaction and mass spectroscopy analysis. Of the 22 radial scars, 14 (63.6%) had PIK3CA mutations (10 with H1047R mutations, 2 G1049R mutations, 1 E542K, 1 E545K). The remaining 8 lesions were wild type for all of the screened genes. Of the radial scars without epithelial atypia, 9/16 (56.3%) had PIK3CA mutations; furthermore, 5/6 (83.3%) radial scars with atypia had mutations detected. In this study, the frequency of PIK3CA mutations was notably higher than the 25% to 30% mutation frequency of invasive breast cancer. This finding raises interesting questions as to the role of PIK3CA mutations in breast cancer development. Additional larger studies are indicated to confirm and extend these observations in understanding the pathogenesis of radial scars and their relationship to breast cancer.

Abstract B38: Evaluation of the OnTarget™ system for highly sensitive mutation detection.

Abstract Background: Effective non-invasive methods for patient characterization and monitoring during treatment are desired. Cell-free circulating tumor DNA in plasma is elevated in cancer patients, and can be a valuable tool for evaluating mutation profile over time. Low mutant allele fraction in a limited amount of low quality DNA makes it very challenging to accurately characterize plasma samples. Therefore, a highly sensitive and robust testing method is required for genetic characterization of circulating tumor DNA. The goals of this study were to: • Evaluate assay performance and limit of detection of the Boreal Genomics OnTarget™ system 18-mutation panel using human germline DNA • Assess the relative sensitivity of the OnTarget™ system for KRAS G13D and PIK3CA H1047R mutation detection using cell line derived DNA • Compare assay performance and sensitivity with multiple DNA input amounts Materials and methods: DNA was extracted from normal healthy human blood and HCT116 (KRAS G13D and PIK3CA H1047R heterozygous mutant) cell line pellets. The cell line DNA was diluted with the human blood DNA to produce samples with known ratios of mutant and wild-type alleles. Samples were run with the OnTarget™ mutation detection technology platform 18-mutation panel. The platform utilizes oligo probes and a gel based system to enrich targeted mutant fragments from a DNA sample, followed by PCR amplification and next generation sequencing with the Illumina MiSeq™ platform. The 18-plex panel tests mutant allele percentage of 18 common cancer mutations in the BRAF, EGFR, KRAS, and PIK3CA genes. Results: Repeat testing of germline DNA from multiple human donors was conducted to determine the background variability (noise) of the 18-plex assay. The mean mutant allele percentage for 10 of the 18 mutations assessed was under 0.001%. 7 of the mutations were under 0.01%, with one other mutation was just over 0.01%. Comparing mutant allele percentages of the titrated cell line DNA samples assessed limit of detection of two of the assays at the 300ng input level. The PIK3CA H104R assay detected mutant DNA over the background levels at the 0.01 and 0.001% input amount of mutant DNA. The KRAS G13D assay signal was over background at the 0.01% input, but within the assay noise at the 0.001% input. When testing the same samples at 30ng of DNA input, all mutation detection was similar, except the PIK3CA mutation was not detected at the 0.001% input amount. Summary: The OnTarget™ mutation enrichment system followed by NGS provides high sensitivity mutation profiling of DNA with high multiplexing capabilities. The low DNA input requirement and limit of detection make it a useful platform for testing of cell-free circulating plasma DNA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B38. Citation Format: Russell M. Walker, Sara Dunaj, Dalin Chan, Sunita Badola. Evaluation of the OnTarget™ system for highly sensitive mutation detection. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B38.

Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity

Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53-MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53-MDMX interaction, SJ-172550, the efficacy of a simplified yeast-based screening assay to search for inhibitors of p53-MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild-type (wt) p53-induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53-induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin-α, nutlin-3a and SJ-172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.

A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

BackgroundMutations of the p53 oncosuppressor gene are amongst the most frequent aberration seen in human cancer. Some mutant (mt) p53 proteins are prone to loss of Zn(II) ion that is bound to the wild-type (wt) core, promoting protein aggregation and therefore unfolding. Misfolded p53 protein conformation impairs wtp53-DNA binding and transactivation activities, favouring tumor growth and resistance to antitumor therapies. Screening studies, devoted to identify small molecules that reactivate mtp53, represent therefore an attractive anti-cancer therapeutic strategy. Here we tested a novel fluorescent curcumin-based Zn(II)-complex (Zn-curc) to evaluate its effect on mtp53 reactivation in cancer cells.MethodsP53 protein conformation was examined after Zn-curc treatment by immunoprecipitation and immunofluorescence assays, using conformation-specific antibodies. The mtp53 reactivation was evaluated by chromatin-immunoprecipitation (ChIP) and semi-quantitative RT-PCR analyses of wild-type p53 target genes. The intratumoral Zn-curc localization was evaluated by immunofluorescence analysis of glioblastoma tissues of an ortothopic mice model.ResultsThe Zn-curc complex induced conformational change in p53-R175H and -R273H mutant proteins, two of the most common p53 mutations. Zn-curc treatment restored wtp53-DNA binding and transactivation functions and induced apoptotic cell death. In vivo studies showed that the Zn-curc complex reached glioblastoma tissues of an ortothopic mice model, highlighting its ability to crossed the blood-tumor barrier.ConclusionsOur results demonstrate that Zn-curc complex may reactivate specific mtp53 proteins and that may cross the blood-tumor barrier, becoming a promising compound for the development of drugs to halt tumor growth.

AKT, EGFR, C-ErbB-2, and C-Kit Expression in Uterine Carcinosarcoma

Uterine carcinosarcoma (UCS) accounts for approximately 15% of uterine cancer-associated deaths in the United States. With lack of effective treatment modalities, identification of underlying molecular defects may allow the introduction of targeted treatments. The expression of AKT, epithelial growth factor receptor, C-Kit, and C-ErbB-2 were studied by immunohistochemistry and exons 9 and 20 of PIK3CA gene were sequences in a cohort of 37 UCS, including 23 early-stage (I and II) and 14 late-stage (III and IV) tumors. Twenty-three (62%) of the UCS were homologous; the reminder contained heterologous elements. The carcinomatous component was pure serous carcinoma in 13 (35%), endometrioid in 12 (32%) cases. An immunostaining score ranging from 0 to (6+) was calculated for AKT, epithelial growth factor receptor, and C-Kit. C-ErbB-2 staining was scored by American Society of Clinical Oncology/College of American Pathologists criteria. AKT staining was seen in 35/37 cases with an immunostaining score ranging from (2+) to (5+). AKT was expressed significantly more in the early-stage than late-stage disease (P=0.016). The expression of AKT in the epithelial component was associated with the survival (P=0.026). Epithelial growth factor receptor was positive in 21/37 cases. Only 8 cases showed (≤3+) immunostaining score with C-Kit. C-ErbB-2 immunostain was (3+) in only 1 case. An H1047R mutation on PIK3CA gene was detected in both carcinomatous and sarcomatous components in a single case. These results indicate that AKT pathway may be important in pathogenesis of UCS. Further studies with larger cohorts are warranted to confirm the observed associations in this study.

Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin

Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

PIK3CA gene mutation associated with poor prognosis of lung adenocarcinoma

PurposePIK3CA gene mutations have been detected in many malignancies, but the frequency of different mutations and their role in the carcinogenesis of lung adenocarcinoma are still unclear. The purpose of this study was to explore the clinical pathological impact and prognostic implications of PIK3CA mutations in lung adenocarcinoma.MethodsFive common PIK3CA mutations (E542K, E545K, and E545D mutation in exon 9, H1047R and H1047L mutation in exon 20) were detected by amplification refractory mutation system (ARMS) allele-specific polymerase chain reaction (PCR), in 122 patients with lung adenocarcinoma. The relationships were studied between these mutations and various clinicopathologic variables (age, lymph node status, distant metastasis, clinicopathologic stage, smoking status, and progression-free survival).ResultsIn total, 25 mutations were identified, of which 24 mutations were clustered in exon 20, and one mutation in exon 9. The most common mutations were H1047R (18 out of the 122 patients, 14.8%) in exon 20. PIK3CA-mutated tumors were more frequently found in patients with lymph node positive metastasis status (P < 0.05). There was no significant association between PIK3CA mutations and age, distant metastasis, smoking status, or clinicopathologic stage. However, mutations were found less frequently in the early clinicopathologic stage patients (six in 50 cases, 12%) than in advanced stage (19 in 72 cases, 26.4%). Higher frequency of H1047R mutations was associated with poor prognosis, and this association reached statistical significance (P < 0.05).ConclusionOur data indicate that the PIK3CA mutations H1047R and H1047L are significant genetic alterations in lung adenocarcinoma. Among lung adenocarcinoma patients who underwent curative resection, PIK3CA mutations were associated with shorter progression-free survival. Our findings demonstrated a significant role of PIK3CA in lung adenocarcinoma.

Gliosarcoma arising from an oligodendroglioma (oligosarcoma)

Gliosarcoma, a biphasic tumor with both mesenchymal and glial elements, is typically considered a variant of astrocytoma (glioblastoma), WHO Grade IV. A 57-year-old man presented with altered mental status and was found to have a large right frontal mass. Biopsy and subsequent subtotal resection revealed a WHO Grade II oligodendroglioma with classic histological features, expression of IDH1 R132H mutant protein, and chromosome 1p19q co-deletion. Fifteen months later, the patient developed recurrent tumor composed of intersecting fascicles of spindled cells with necrosis and a high mitotic index. The recurrent tumor stained for both mesenchymal and glial elements, consistent with the diagnosis of gliosarcoma, and showed retained IDH1 R132H expression. By FISH analysis, the gliosarcoma showed no evidence of 1p19q co-deletion. We performed SNP arrays and detailed SNP analysis of both the oligodendroglioma and the gliosarcoma. This demonstrated loss of heterozygosity (LOH) of chromosomes 1 and 19 in the gliosarcoma with retention of the same full-length chromosomes 1 and 19 found intact in the oligodendroglioma. Not surprisingly, the gliosarcoma harbored multiple additional alterations, consistent with clonal evolution. There have been only rare reports of sarcomatous transformation of oligodendroglioma ("oligosarcoma") and most were published prior to the development of modern genetic modalities. Here we present a case with detailed genetic evidence that suggests that mesenchymal metaplasia sarcomatous transformation is possible in classic oligodendrogliomas with 1p19q codeletions.