We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.
Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein-protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable.
Sickle cell disease (SCD) is an inherited blood disorder that affects about 100,000 people in the U.S., primarily Blacks/African-Americans. A multitude of complications negatively impacts quality of life. Hydroxyurea has been FDA approved since 1998 as a disease-modifying therapy for SCD, but is underutilized. Negative and uninformed perceptions of hydroxyurea and barriers to its use hinder adherence and promotion of the medication. As the largest real-world study to date that assessed hydroxyurea use for children and adults with SCD, we gathered and analyzed perspectives of providers, individuals with SCD, and families. Participants provided information about socio-demographics, hospital and emergency admissions for pain, number of severe pain episodes interfering with daily activities, medication adherence, and barriers to hydroxyurea. Providers reported on indications for hydroxyurea, reasons not prescribed, and current laboratory values. We found that hydroxyurea use was reported in over half of eligible patients from this large geographic region in the U.S., representing a range of sickle cell specialty clinical settings and practices. Provider and patient/caregiver reports about hydroxyurea use were consistent with one another; adults 26 years and older were least likely to be on hydroxyurea; and the likelihood of being on hydroxyurea decreased with one or more barriers. Using the intentional and unintentional medication nonadherence framework, we found that, even for patients on hydroxyurea, challenges to taking the medicine at the right time and forgetting were crucial unintentional barriers to adherence. Intentional barriers such as worry about side effects and “tried and it did not work” were important barriers for young adults and adults. For providers, diagnoses other than HgbSS or HgbS-β0 thalassemia were associated with lower odds of prescribing, consistent with evidence-based guidelines. Our results support strengthening provider understanding and confidence in implementing existing SCD guidelines, and the importance of shared decision making. Our findings can assist providers in understanding choices and decisions of families; guide individualized clinical discussions regarding hydroxyurea therapy; and help with developing tailored interventions to address barriers. Addressing barriers to hydroxyurea use can inform strategies to minimize similar barriers in the use of emerging and combination therapies for SCD.
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, H104R 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 (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, III, Peter K. Vogt. Butterfly effect in cancer: How a PI3-kinase mutation remodels the cell. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A28.
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