Michèl Schummer

Proteomic analysis of ovarian cancer cells reveals dynamic processes of protein secretion and shedding of extra-cellular domains

Background Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid. Methodology and Findings To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [13C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions. Conclusions and Significance Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.

Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the established marker CA125.

OBJECTIVEnEvaluate and compare the effectiveness of CA125, HE4, Mesothelin and MMP7 marker levels to monitor ovarian cancer patients after surgery and chemotherapy. Evaluate the lead time of a rise of marker levels before recurrence.nnnMETHODSnThe study consists of 23 patients with advanced stage ovarian/fallopian tube cancer. Blood was drawn after front line surgery and chemotherapy treatment and at 3 month intervals thereafter. One patient had chemoresistant disease, two patients remained in remission and 20 patients had recurring disease and were used for marker evaluation.nnnRESULTSnIn five patients HE4 was the only marker to elevate before recurrence with a lead time of up to 4½ months including one patient who did not have a CA125 response at all. In a further two patients, HE4 increased before CA125 did. In four of these seven patients, HE4 levels were consistently at or above threshold during remission when both CA125 and imaging results were negative. MMP7 elevated before recurrence in one patient who was negative for the other markers. Mesothelin elevated in two patients who were also positive for CA125 and HE4.nnnCONCLUSIONSnHE4 can predict ovarian cancer recurrence earlier than CA125 and it can be elevated in patients that do not express CA125 at sufficient levels to make a clinical decision. MMP7 and Mesothelin have lower potential as markers for ovarian cancer recurrence to complement CA125. A failure of HE4 levels to normalize at completion of standard therapy may indicate a poor prognosis.

Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers

Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) down-regulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.

Comparison of Breast Cancer to Healthy Control Tissue Discovers Novel Markers with Potential for Prognosis and Early Detection

This study was initiated to identify biomarkers with potential value for the early detection of poor-outcome breast cancer. Two sets of well-characterized tissues were utilized: one from breast cancer patients with favorable vs. poor outcome and the other from healthy women undergoing reduction mammaplasty. Over 46 differentially expressed genes were identified from a large list of potential targets by a) mining publicly available expression data (identifying 134 genes for quantitative PCR) and b) utilizing a commercial PCR array. Three genes show elevated expression in cancers with poor outcome and low expression in all other tissues, warranting further investigation as potential blood markers for early detection of cancers with poor outcome. Twelve genes showed lower expression in cancers with poor outcome than in cancers with favorable outcome but no differential expression between aggressive cancers and most healthy controls. These genes are more likely to be useful as prognostic tissue markers than as serum markers for early detection of aggressive disease. As a secondary finding was that, when histologically normal breast tissue was removed from a distant site in a breast with cancer, 7 of 38 specimens displayed a cancer-like expression profile, while the remaining 31 were genetically similar to the reduction mammaplasty control group. This finding suggests that some regions of ipsilateral histologically ‘normal’ breast tissue are predisposed to becoming malignant and that normal-appearing tissue with malignant signature might warrant treatment to prevent new primary tumors.

Breast cancer genomics: normal tissue and cancer markers

Mammography is a powerful screening tool for early detection of breast cancer, but it has limitations in terms of both specificity and sensitivity. Imaging tools such as MRI that complement mammography are too costly to serve as first‐line screens. Recently, progress has been made on blood markers, particularly microRNAs and proteins. There are new methods for protein marker discovery directly in blood, but they are limited in the number of patients that can be examined. An alternative is to discover markers as transcripts in tissues, followed by development of blood protein tests for those that perform best. To identify genes that are overexpressed in malignancy it is paramount to include normal control tissues from healthy individuals. Here we report the identification of potential breast cancer markers, including some that are overexpressed in aggressive disease.

Evaluating Serum Markers for Hormone Receptor-Negative Breast Cancer

Introduction Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Death rates have been declining, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of breast disease that might not require treatment, and under-diagnosis of cancer in women with dense breasts. Breast cancer screening could be improved by pairing mammography with a tumor circulating marker, of which there are currently none. Given genomic similarities between the basal breast cancer subtype and serous ovarian cancer, and given our success in identifying circulating markers for ovarian cancer, we investigated the performance in hormone receptor-negative breast cancer detection of both previously identified ovarian serum markers and circulating markers associated with transcripts that were differentially expressed in breast cancer tissue compared to healthy breast tissue from reduction mammaplasties. Methods We evaluated a total of 15 analytes (13 proteins, 1 miRNA, 1 autoantibody) in sera drawn at or before breast cancer surgery from 43 breast cancer cases (28 triple-negative—TN—and 15 hormone receptor-negative—HRN—/ HER2-positive) and 87 matched controls. Results In the analysis of our whole cohort of breast cancer cases, autoantibodies to TP53 performed significantly better than the other selected 14 analytes showing 25.6% and 34.9% sensitivity at 95% and 90% specificity respectively with AUC: 0.7 (p<0.001). The subset of 28 TN cancers showed very similar results. We observed no correlation between anti-TP53 and the 14 other markers; however, anti-TP53 expression correlated with Body-Mass-Index. It did not correlate with tumor size, positive lymph nodes, tumor stage, the presence of metastases or recurrence. Conclusion None of the 13 serum proteins nor miRNA 135b identified women with HRN or TN breast cancer. TP53 autoantibodies identified women with HRN breast cancer and may have potential for early detection, confirming earlier reports. TP53 autoantibodies are long lasting in serum but may be affected by storage duration. Autoantibodies to TP53 might correlate with Body-Mass-Index.

Abstract 586: Genomic and functional characterizations of phosphodiesterase 4Din human cancers.

Background Phosphodiesterase 4D (PDE4D) degrades the secondary messenger adenosine 3’, 5’-cyclic monophosphate (cAMP). PDE4D recently was implicated in cancer cell proliferation, but alterations in its genome and function in tumors are unclear. Methods PDE4D gene copy number determined by SNP-CHIP was comprehensively analyzed in a large number of solid tumor specimens from diverse origins (n=5,569). Expression of PDE4D gene transcripts were examined in 971 primary tumors, as well as established cancer cell lines. PDE4D protein expression in 11 different types of cancers were evaluated by immunohistochemistry (n=165). Endogenous PDE4D in multiple types of tumor cell lines was suppressed by both shRNAs and compound 26B, a novel inhibitor of PDE4D, and the effects on apoptosis and proliferation were examined. We further investigated the molecular mechanisms involved. Effect of ectopic expression of PDE4D on tumor growth was assessed both in vitro and in vivo. Results Homozygous deletion of PDE4D occurred in 198 cases out of 5,569 primary solid tumors (3.56%), with most of them being internal microdeletions. Unexpectedly, these internal microdeletions did not result in loss of its gene products, as the expression of PDE4D mRNA and protein was not compromised in the PDE4D-microdeleted samples. Immunohistochemistry staining revealed that PDE4D protein levels were up-regulated in several types of tumors. Importantly, genetic and pharmacological suppression of endogenous PDE4D caused apoptosis and growth inhibition preferentially in a variety of cancer cells but not in non-malignant epithelial cells. We further showed that anti-tumor events triggered by PDE4D-suppression were lineage-dependently associated with induction of BIM and down-regulation of MITF. Moreover, ectopic expression of the PDE4D short isoform (PDE4D2) enhanced the proliferation of melanoma both in vitro and in vivo. Conclusions We show for the first time that although targeted by genomic homozygous microdeletions, alternatively spliced PDE4D can function as a tumor-promoting factor. PDE4D represents a novel, targetable enzyme of cancer cells. Citation Format: Dechen Lin, Liang Xu, Ling-Wen Ding, Arjun Sharma, Li-Zhen Liu, Henry Yang, Patrick Tan, Jay Vadgama, Beth Karlan, Jenny Lester, Nicole Urban, Michel Schummer, Ngan Doan, Jonathan Said, Martin Walsh, Paresma Patel, Craig Thomas, Daniel Chan, Phillip Koeffler. Genomic and functional characterizations of phosphodiesterase 4Din human cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 586. doi:10.1158/1538-7445.AM2013-586

Breast cancer genomics

Mammography is a powerful screening tool for early detection of breast cancer, but it has limitations in terms of both specificity and sensitivity. Imaging tools such as MRI that complement mammography are too costly to serve as first‐line screens. Recently, progress has been made on blood markers, particularly microRNAs and proteins. There are new methods for protein marker discovery directly in blood, but they are limited in the number of patients that can be examined. An alternative is to discover markers as transcripts in tissues, followed by development of blood protein tests for those that perform best. To identify genes that are overexpressed in malignancy it is paramount to include normal control tissues from healthy individuals. Here we report the identification of potential breast cancer markers, including some that are overexpressed in aggressive disease.