Emmanuel F. Petricoin

Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer

Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA‐125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI‐FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two‐dimensional gel electrophoresis (2‐D PAGE) to identify proteins that may serve as invasive ovarian cancer‐specific biomarkers for early detection and/or new therapeutic targets. We performed 2‐D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2‐D gels created from microdissected lysates with SYPRO‐Ruby stained 2‐D PAGE profiles of the patient‐matched undissected bulk tumor lysates from all five patients. Twenty‐three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby‐red stained gels was performed by LCM coupled Western blot and reverse‐phase array technology in a study set of six cases (the aforementioned five cases used in the 2‐D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G‐protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Cancer diagnosis using proteomic patterns

The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility and monitor progression. Much of this effort has focused upon the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian cancer. Proteomic pattern analysis relies on the pattern of proteins observed and does not rely on the identification of a traceable biomarker. Hundreds of clinical samples per day can be analyzed utilizing this technology, which has the potential to be a novel, highly sensitive diagnostic tool for the early detection of cancer.

Sensitive Immunoassay of Tissue Cell Proteins Procured by Laser Capture Microdissection

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Genomics and proteomics: application of novel technology to early detection and prevention of cancer

Advances in molecular biology over the past decade have helped to enhance our understanding of the complex interplay between genetic, transcriptional and translational alterations in human cancers. These molecular changes are the basis for an evolving field of high-throughput cancer discovery techniques using microscopic amounts of patient-based materials. Laser capture microdissection allows pure populations of cells to be isolated from both the tumor and stroma in order to identify subtle differences in RNA and protein expression. Comparative analysis of these alterations between normal, pre-invasive, and invasive tissue using powerful bioinformatics programs has allowed us to identify novel tumor markers, profile complex protein pathways, and develop new molecular-based therapies. Continued refinement of such high-throughput microtechnologies will enable us to rapidly query patient specimens to identify novel methods for early detection, treatment, and follow-up of a wide array of human cancers.

Signal pathway profiling of epithelial and stromal compartments of colonic carcinoma reveals epithelial-mesenchymal transition

Molecular crosstalk, including reciprocal stimulation, is theorized to take place between epithelial cancer cells and surrounding non-neoplastic stromal cells. This is the rationale for stromal therapy, which could eliminate support of a cancer by its genetically stable stroma. Epithelial-stromal crosstalk is so far poorly documented in vivo, and cell cultures and animal experiments may not provide accurate models. The current study details stromal-epithelial signalling pathways in 35 human colon cancers, and compares them with matched normal tissues using quantitative proteomic microarrays. Lysates prepared from separately microdissected epithelium and stroma were analysed using antibodies against 61 cell signalling proteins, most of which recognize activated phospho-isoforms. Analyses using unsupervised and supervised statistical methods suggest that cell signalling pathway profiles in stroma and epithelium appear more similar to each other in tumours than in normal colon. This supports the concept that coordinated crosstalk occurs between epithelium and stroma in cancer and suggests epithelial-mesenchymal transition. Furthermore, the data herein suggest that it is driven by cell proliferation pathways and that, specifically, several key molecules within the mitogen-activated protein kinase pathway may play an important role. Given recent findings of epithelial-mesenchymal transition in therapy-resistant tumour epithelium, these findings could have therapeutic implications for colon cancer.

Application of molecular technologies for phosphoproteomic analysis of clinical samples

The integration of small kinase inhibitors and monoclonal antibodies into oncological practice has opened a new paradigm for treating cancer patients. As proteins are the direct targets of the new generations of targeted therapeutics, many of which are kinase/enzymatic inhibitors, there is an increasing interest in developing technologies capable of monitoring post-translational changes of the human proteome for the identification of new predictive, prognostic and therapeutic biomarkers. It is well known that the vast majority of the activation/deactivation of these drug targets is driven by phosphorylation. This review provides a description of the main proteomic platforms (planar and bead array, reverse phase protein microarray, phosphoflow, AQUA and mass spectrometry) that have successfully been used for measuring changes in phosphorylation level of drug targets and downstream substrates using clinical specimens. Major emphasis was given to the strengths and weaknesses of the different platforms and to the major barriers that are associated with the analysis of the phosphoproteome. Finally, a number of examples of application of the above-mentioned technologies in the clinical setting are reported.

CXCR4 heterogeneity in primary cells: possible role of ubiquitination

The chemokine receptor CXCR4 is a primary coreceptor for the HIV‐1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45–47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T‐lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti‐CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two‐dimensional gels, antiubiquitin antibodies reacted with the 62‐kDa CXCR4 species from monocytes subsequent to coprecipitation with anti‐CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101‐kDa CXCR4 isoform in immunoblots by three‐ to sevenfold. In lymphocytes, lactacystin also increased cell‐surface expression of CXCR4, which correlated with enhanced fusion with HIV‐1 envelope‐expressing cells. Similar increases in the intensity of the 101‐kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti‐CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.

Acquisition of estrogen independence induces TOB1-related mechanisms supporting breast cancer cell proliferation.

Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, a systems biology approach was used. A library of small interfering RNAs targeting an estrogen receptor (ER)- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent ER-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mammalian target of rapmycin (mTOR) inhibitors in estrogen-independent cells but not in estrogen-dependent cells. Phosphoproteomic profiles from reverse-phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with ER-positive breast cancers.