A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

Molecular insights into cancer drug resistance from a proteomics perspective

Introduction: Resistance to chemotherapy and development of specific and effective molecular targeted therapies are major obstacles facing current cancer treatment. Comparative proteomic approaches have been employed for the discovery of putative biomarkers associated with cancer drug resistance and have yielded a number of candidate proteins, showing great promise for both novel drug target identification and personalized medicine for the treatment of drug-resistant cancer.

Areas covered: Herein, we review the recent advances and challenges in proteomics studies on cancer drug resistance with an emphasis on biomarker discovery, as well as understanding the interconnectivity of proteins in disease-related signaling pathways. In addition, we highlight the critical role that post-translational modifications (PTMs) play in the mechanisms of cancer drug resistance.

Expert opinion: Revealing changes in proteome profiles and the role of PTMs in drug-resistant cancer is key to deciphering the mechanisms of treatment resistance. With the development of sensitive and specific mass spectrometry (MS)-based proteomics and related technologies, it is now possible to investigate in depth potential biomarkers and the molecular mechanisms of cancer drug resistance, assisting the development of individualized therapeutic strategies for cancer patients.     

Comparative proteomic analysis of cisplatin sensitive IGROV1 ovarian carcinoma cell line and its resistant counterpart IGROV1-R10

Ovarian cancer is one of the leading causes of mortality due to gynaecological cancer. Despite a good response to surgery and initial chemotherapy essentially based on cisplatin (cis-diamino-dichloro-platinum(II) (CDDP)) compounds, late tumour detection and frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been implicated in CDDP resistance but they are not sufficient to exhaustively explain this resistance emergence. We applied a proteomic approach based on 2-DE coupled with MS to identify proteins associated with the chemoresistance process. We first established a proteomic pattern of the CDDP sensitive ovarian cell line IGROV1 using MALDI-TOF-MS and PMF. We then compared this 2-D pattern with that of the CDDP-resistant counterpart IGROV1-R10. Among the 40 proteins identified, cytokeratins 8 and 18 and aldehyde dehydrogenase 1 were overexpressed in IGROV1-R10, whereas annexin IV was down-regulated. These observations have been confirmed by Western blotting. The characterization of such variations could lead to the development of new protein markers or to the establishment of new therapeutic strategies. Moreover, the identification of proteins involved in CDDP resistance in ovarian tumours would be useful in completing our understanding on this complex mechanism.     

Systematic identification of dysregulated lncRNAs associated with platinum-based chemotherapy response across 11 cancer types

Aberrant expression of long non-coding RNAs (lncRNAs) leads to the development of chemoresistance by regulating a series of biological processes, which is one of the major obstacles in the cancer treatment. This study aimed to identify some key lncRNAs that are associated with platinum-based chemoresistance in multiple cancers. Regulating the expression levels of these lncRNAs can enhance the sensitivity of patients to chemotherapy drugs and improve the therapeutic effect of cancer. By systematically analyzing 648 samples regarding platinum drug response from the Cancer Genome Atlas (TCGA), we have identified 32 dysregulated lncRNAs across 11 cancer types that could affect platinum-based chemotherapy response, of which 78.125% (25/32) were significantly down-regulated in drug-resistant samples. Drug response prediction model that had been constructed based on the expression pattern of these dysregulated lncRNAs could accurately predict the chemotherapy response of tumor patients, and the area under the curve (AUC) was between 0.8034 and 0.9984. In particular, all of these dysregulated lncRNAs that we identified were cancer-specific. They were significantly associated with the survival of tumor patients and could serve as cancer-specific biomarkers for prognosis. In conclusion, this study will contribute to improving the drug resistance of tumor patients during chemotherapy, and it is of real significance for selecting effective chemotherapy drugs and achieving precision medicine.     

iTRAQ-based proteomic profiling of breast cancer cell response to doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC-MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer.     

Proteomic profiling of pancreatic cancer for biomarker discovery

Pancreatic cancer is a uniformly lethal disease that is difficult to diagnose at early stage and even more difficult to cure. In recent years, there has been a substantial interest in applying proteomics technologies to identify protein biomarkers for early detection of cancer. Quantitative proteomic profiling of body fluids, tissues, or other biological samples to identify differentially expressed proteins represents a very promising approach for improving the outcome of this disease. Proteins associated with pancreatic cancer identified through proteomic profiling technologies could be useful as biomarkers for the early diagnosis, therapeutic targets, and disease response markers. In this article, we discuss recent progress and challenges for applying quantitative proteomics technologies for biomarker discovery in pancreatic cancer.     

Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.     

Novel pathways involved in cisplatin resistance identified by a proteomics approach in non-small-cell lung cancer cells

Although platinum-based chemotherapy remains the standard-of-care for most patients with advanced non-small-cell lung cancer (NSCLC), acquired resistance occurs frequently predicting poor prognosis. To examine the mechanisms underlying platinum resistance, we have generated and characterized by proteomic approach the resistant A549 CDDP-resistant (CPr-A549) and their parental A549 cells, identifying 15 proteins differentially expressed (13 upregulated and 2 downregulated in CPr-A549). In details, we highlighted a coherent network of proteins clustering together and involved in altered protein folding and endoplasmic reticulum stress, correlated with epithelial to mesenchymal transition process and cancer stem cell markers, where vimentin played a hierarchical role, ultimately resulting in increased aggressive features. By using publicly available databases we showed that the modulated proteins could contribute to NSCLC carcinogenesis and correlate with NSCLC patients prognosis and survival probability, suggesting that they can be used as novel potential prognostic/predictive biomarkers or therapeutic targets to overcome platinum-resistance.     

A proteomic kinetic analysis of IGROV1 ovarian carcinoma cell line response to cisplatin treatment

Ovarian cancer is one of the leading causes of mortality by gynecological cancer. Despite good response to surgery and initial chemotherapy, essentially based on cisplatin (cis-diamino-dichloro-platinum(II) (CDDP)) compounds, frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been described as implicated in CDDP resistance, however they are not sufficient to exhaustively account for this resistance emergence. We applied a proteomic approach based on 2-DE coupled with MS (MALDI-TOF/TOF) to identify proteins associated with chemoresistance induced by CDDP. A kinetic analysis of IGROV1 cell behavior following treatment with CDDP and subsequent statistical analysis revealed time and/or concentration-dependent modifications in protein expression. We evidenced events such as decreased amino-acid and nucleotide synthesis potentially associated with cell cycle blockade, and variations that may be related to resistance acquisition, such as possible enhanced glycolysis and increased proliferating potential. Moreover, overexpressions of aldehyde dehydrogenase 1 and both cytokeratins 8 and 18 were consistent with our previous findings, demonstrating that expression of these proteins was increased in cisplatin-resistant IGROV1-R10 as compared to IGROV1 parental cells. Identification of such proteins could allow improved understanding of the mechanisms leading to cell death or survival and, thus, to the acquisition of chemoresistance.     

Discovery of IL-18 as a novel secreted protein contributing to doxorubicin resistance by comparative secretome analysis of MCF-7 and MCF-7/Dox

Background

Resistance to chemotherapy is the major cause of failure in breast cancer treatment. Recent studies suggest that secreted proteins may play important roles in chemoresistance. We sought to systematically characterize secreted proteins associated with drug resistance, which may represent potential serum biomarkers or novel drug targets.

Methodology/Principal Findings

In the present work, we adopted the proteomic strategy of one-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry to compare the secretome of MCF-7 and doxorubicin-resistant MCF-7/Dox. A total of 2,084 proteins were identified with at least two unique peptides in the conditioned media of two cell lines. By quantification with label-free spectral counting, 89 differentially expressed secreted proteins (DESPs) between the two cell lines were found. Among them, 57 DESPs were first found to be related to doxorubicin resistance in this work, including 24 extracellular matrix related proteins, 2 cytokines and 31 unclassified proteins. We focused on 13 novel DESPs with confirmed roles in tumor metastasis. Among them, the elevated expression of IL-18 in doxorubicin-resistant cell lines and breast tumor tissues was validated and its role in doxorubicin resistance was further confirmed by cell viability experiments in the presence or absence of this protein.

Conclusions/Significance

Comparative analysis of the secretome of MCF-7 and MCF-7/Dox identified novel secreted proteins related to chemotherapy resistance. IL-18 was further validated to contribute to doxorubicin resistance, in addition to its confirmed role in breast cancer metastasis. Due to its dual roles in both drug resistance and tumor metastasis, IL-18 may represent a useful drug target for breast cancer therapy.     

Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines

Chemoresistance is a major therapeutic obstacle in cancer patients, and the mechanisms of drug resistance are not fully understood. In the present study, we established platinum-resistant human ovarian cancer cell lines and identified differentially expressed proteins related to platinum resistance. The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP) human ovarian cancer cell lines were isolated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In total, 57 differential protein spots were identified, and five proteins, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1 (GSTO1-1), and cytosolic NADP+-dependent isocitrate dehydrogenase (IDHc), were found to be co-instantaneous significance compared with their parental cells. The expression of the five proteins was validated by quantitative PCR and western blot, and the western blot results showed complete consistency with proteomic techniques. The five proteins are hopeful to become candidates for platinum resistance. These may be useful for further study of resistance mechanisms and screening of resistant biomarkers.     

Proteomic approach reveals FKBP4 and S100A9 as potential prediction markers of therapeutic response to neoadjuvant chemotherapy in patients with breast cancer

Although doxorubicin (Doxo) and docetaxel (Docet) in combination are widely used in treatment regimens for a broad spectrum of breast cancer patients, a major obstacle has emerged in that some patients are intrinsically resistant to these chemotherapeutics. Our study aimed to discover potential prediction markers of drug resistance in needle-biopsied tissues of breast cancer patients prior to neoadjuvant chemotherapy. Tissues collected before chemotherapy were analyzed by mass spectrometry. A total of 2,331 proteins were identified and comparatively quantified between drug sensitive (DS) and drug resistant (DR) patient groups by spectral count. Of them, 298 proteins were differentially expressed by more than 1.5-fold. Some of the differentially expressed proteins (DEPs) were further confirmed by Western blotting. Bioinformatic analysis revealed that the DEPs were largely associated with drug metabolism, acute phase response signaling, and fatty acid elongation in mitochondria. Clinical validation of two selected proteins by immunohistochemistry found that FKBP4 and S100A9 might be putative prediction markers in discriminating the DR group from the DS group of breast cancer patients. The results demonstrate that a quantitative proteomics/bioinformatics approach is useful for discovering prediction markers of drug resistance, and possibly for the development of a new therapeutic strategy.     

Role of ABCB1 in mediating chemoresistance of triple-negative breast cancers

Triple-negative breast cancer (TNBC) is a group of breast cancers which neither express hormonal receptors nor human epidermal growth factor receptor. Hence, there is a lack of currently known targeted therapies and the only available line of systemic treatment option is chemotherapy or more recently immune therapy. However, in patients with relapsed disease after adjuvant or neoadjuvant therapy, resistance to chemotherapeutic agents has often developed, which results in poor treatment response. Multidrug resistance (MDR) has emerged as an important mechanism by which TNBCs mediate drug resistance and occurs primarily due to overexpression of ATP-binding cassette (ABC) transporter proteins such as P-glycoprotein (Pgp). Pgp overexpression had been linked to poor outcome, reduced survival rates and chemoresistance in patients. The aim of this mini-review is to provide a topical overview of the recent studies and to generate further interest in this critical research area, with the aim to develop an effective and safe approach for overcoming Pgp-mediated chemoresistance in TNBC.     

A serum protein profile predictive of the resistance to neoadjuvant chemotherapy in advanced breast cancers

Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies.     

Proteomics in pancreatic cancer research

In this review, we give an overview of the actual role of proteomic technologies in the study of pancreatic cancers (PCs). We describe PC proteomics on the basis of sample origins, i.e. tissues, body fluids, and PC cell lines. As regards PC tissues, we report the identification of a number of candidate biomarkers of precursor lesions that may allow early diagnosis of this neoplasia. Moreover, we describe cytoskeletal and hypoxia-regulated proteins that confirm the involvement of cytoskeleton modifications and metabolism adaptations in carcinogenesis. We also discuss the most important biomarkers identified by proteomic analysis involved in local invasion and distant metastasis, and in the cross-talk between pancreatic tumor and the surrounding stroma. Furthermore, we report novel candidate biomarkers identified in serum, plasma, and pancreatic juice of cancer patients compared with cancer-free controls. Proteomic alterations in PC cell line models as compared to normal controls and studies on cell lines treated with drugs or new agents to understand their mechanism of pharmacological action or the onset of drug resistance are also presented. Finally, we discuss the recent improvements obtained in classical 2-DE and high-throughput proteomic strategies able to allow the overcoming of relevant proteomic drawbacks.     

Proteomics discovery of chemoresistant biomarkers for ovarian cancer therapy

Introduction: Chemoresistance is a major challenge to current ovarian cancer chemotherapy. It is important to identify biomarkers to distinguish chemosensitive and chemoresistant patients.

Areas covered: We review the medical literature, discuss MS-based technologies with respect to chemoresistant ovarian cancer and summarize the promising chemoresistant biomarkers identified. In addition, the challenges and future perspectives of biomarker discovery research are explored. With the employment of mass spectrometry-based (MS-based) proteomics, biomarker discovery of ovarian cancer has made great progress in the last decade. Many potential biomarkers were identified by MS-based proteomics technologies, some of which have been validated for further extensive studies in clinical settings.

Expert commentary: The discovery of chemoresistant biomarkers is a newly developing area and may provide a clue for predicting chemotherapeutic response and discover therapeutic targets for paving the way of personalized medicine. Multiple complementary MS-based proteomics approaches hold promise for finding novel therapeutic targets in ovarian cancer treatment.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Proteomics approach to identify biomarkers for upper gastrointestinal cancer

Introduction: The prognosis for patients with upper gastrointestinal cancers remains dismal despite the development of multimodality therapies that incorporate surgery, chemotherapy, and radiotherapy. Early diagnosis and personalized treatment should lead to better prognosis. Given the advances in proteomic technologies over the past decades, proteomics promises to be the most effective technique to identify novel diagnostics and therapeutic targets.

Areas covered: For this review, keywords were searched in combination with ‘proteomics’ and ‘gastric cancer’ or ‘esophageal cancer’ in PubMed. Studies that evaluated proteomics associated with upper gastrointestinal cancer were identified through reading, with several studies quoted at second hand. We summarize the proteomics involved in upper gastrointestinal cancer and discuss potential biomarkers and therapeutic targets.

Expert commentary: In particular, the development of mass spectrometry has enabled detection of multiple proteins and peptides in more biological samples over a shorter time period and at lower cost than was previously possible. In addition, more sophisticated protein databases have allowed a wider variety of proteins in samples to be quantified. Novel biomarkers that have been identified by new proteomic technologies should be applied in a clinical setting.     

Tumour suppressor genes in chemotherapeutic drug response

Since cancer is one of the leading causes of death worldwide, there is an urgent need to find better treatments. Currently, the use of chemotherapeutics remains the predominant option for cancer therapy. However, one of the major obstacles for successful cancer therapy using these chemotherapeutics is that patients often do not respond or eventually develop resistance after initial treatment. Therefore identification of genes involved in chemotherapeutic response is critical for predicting tumour response and treating drug-resistant cancer patients. A group of genes commonly lost or inactivated are tumour suppressor genes, which can promote the initiation and progression of cancer through regulation of various biological processes such as cell proliferation, cell death and cell migration/invasion. Recently, mounting evidence suggests that these tumour suppressor genes also play a very important role in the response of cancers to a variety of chemotherapeutic drugs. In the present review, we will provide a comprehensive overview on how major tumour suppressor genes [Rb (retinoblastoma), p53 family, cyclin-dependent kinase inhibitors, BRCA1 (breast-cancer susceptibility gene 1), PTEN (phosphatase and tensin homologue deleted on chromosome 10), Hippo pathway, etc.] are involved in chemotherapeutic drug response and discuss their applications in predicting the clinical outcome of chemotherapy for cancer patients. We also propose that tumour suppressor genes are critical chemotherapeutic targets for the successful treatment of drug-resistant cancer patients in future applications.