Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid.

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

Proteomic-based biosignatures in breast cancer classification and prediction of therapeutic response.

Protein-based markers that classify tumor subtypes and predict therapeutic response would be clinically useful in guiding patient treatment. We investigated the LC-MS/MS-identified protein biosignatures in 39 baseline breast cancer specimens including 28 HER2-positive and 11 triple-negative (TNBC) tumors. Twenty proteins were found to correctly classify all HER2 positive and 7 of the 11 TNBC tumors. Among them, galectin-3-binding protein and ALDH1A1 were found preferentially elevated in TNBC, whereas CK19, transferrin, transketolase, and thymosin ß4 and ß10 were elevated in HER2-positive cancers. In addition, several proteins such as enolase, vimentin, peroxiredoxin 5, Hsp 70, periostin precursor, RhoA, cathepsin D preproprotein, and annexin 1 were found to be associated with the tumor responses to treatment within each subtype. The MS-based proteomic findings appear promising in guiding tumor classification and predicting response. When sufficiently validated, some of these candidate protein markers could have great potential in improving breast cancer treatment.

Neutralizing monoclonal antibody to periostin inhibits ovarian tumor growth and metastasis.

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein.

Hydrophobic Proteome Analysis of Triple Negative and Hormone-Receptor-Positive-Her2-Negative Breast Cancer by Mass Spectrometer.

INTRODUCTION: It is widely believed that discovery of specific, sensitive, and reliable tumor biomarkers can improve the treatment of cancer. Currently, there are no obvious targets that can be used in treating triple-negative breast cancer (TNBC). METHODS: To better understand TNBC and find potential biomarkers for targeted treatment, we combined a novel hydrophobic fractionation protocol with mass spectrometry LTQ-orbitrap to explore and compare the hydrophobic sub-proteome of TNBC with another subtype of breast cancer, hormone-receptor-positive-Her2-negative breast cancer (non-TNBC). RESULTS: Hydrophobic sub-proteome of breast cancer is rich in membrane proteins. Hundreds of proteins with various defined key cellular functions were identified from TNBC and non-TNBC tumors. In this study, protein profiles of TNBC and non-TNBC were systematically examined, compared, and validated. We have found that nine keratins are down-regulated and several heat shock proteins are up-regulated in TNBC tissues. Our study may provide insights of molecules that are responsible for the aggressiveness of TNBC. CONCLUSION: The initial results obtained using a combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of these proteins appear promising in the discovery of potential cancer biomarkers and bio-signatures. When sufficiently refined, this approach may prove useful in improving breast cancer treatment.

Hydrophobic Fractionation Enhances Novel Protein Detection by Mass Spectrometry in Triple Negative Breast Cancer.

It is widely believed that discovery of specific, sensitive and reliable tumor biomarkers can improve the treatment of cancer. The goal of this study was to develop a novel fractionation protocol targeting hydrophobic proteins as possible cancer cell membrane biomarkers. Hydrophobic proteins of breast cancer tissues and cell lines were enriched by polymeric reverse phase columns. The retained proteins were eluted and digested for peptide identification by nano-liquid chromatography with tandem mass spectrometry using a hybrid linear ion-trap Orbitrap.Hundreds of proteins were identified from each of these three specimens: tumors, normal breast tissue, and breast cancer cell lines. Many of the identified proteins defined key cellular functions. Protein profiles of cancer and normal tissues from the same patient were systematically examined and compared. Stem cell markers were overexpressed in triple negative breast cancer (TNBC) compared with non-TNBC samples. Because breast cancer stem cells are known to be resistant to radiation and chemotherapy, and can be the source of metastasis frequently seen in patients with TNBC, our study may provide evidence of molecules promoting the aggressiveness of TNBC.The initial results obtained using a combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of these proteins appear promising in the discovery of potential cancer biomarkers. When sufficiently refined, this approach may prove useful for early detection and better treatment of breast cancer.

Tumor proteomic profiling predicts the susceptibility of breast cancer to chemotherapy.

Chemotherapy is often used for breast cancer treatment, but individual outcome varies widely. We hypothesized that tumor proteomic profiles obtained prior to chemotherapy may predict the individual tumor response to treatment. The goal of our study was to explore feasibility of using proteomic profiling to preselect patients for an effective chemotherapeutic regimen. Tumors from 52 patients with T2-T4 breast cancer were prospectively collected before neoadjuvant chemotherapy, and were analyzed using surface-enhanced laser desorption ionization/time of flight (SELDI) mass spectrometry. Mass spectral profiles were obtained from tumors with various sensitivities to chemotherapy. Both non-supervised hierarchical clustering and supervised neural network-based classification approaches were employed to compare the profiles. The first two thirds of the enrolled cases (35) were allocated to a training set to select peaks characteristic of resistant tumors. The candidate peaks were used to develop a predicting rule to evaluate the remaining 17 specimens in the validation set. In the training set, the most prominent differences were found between drug resistant and drug susceptible tumors by non-supervised hierarchical clustering. In the validation set, the supervised classification with the K nearest neighbor (KNN) model correctly classified most tumor responses with an accuracy rate of 92.3% [100% of resistant tumors (4/4), and 84.6% of the tumors with favorable response (11/13)]. In the entire group, a single peak at m/z 16,906 correctly separated 88.9% of the tumors with pathologically complete response, and 91.7% of the resistant tumors. The data suggest that breast cancer protein biomarkers may be used to pre-select patients for optimal chemotherapeutic treatment.

Mass spectrometry (LC-MS/MS) site-mapping of N-glycosylated membrane proteins for breast cancer biomarkers.

Cancer cell membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, cellular secretion or cell lysis, and represent promising candidates for interrogation. Because many known disease biomarkers are both glycoproteins and membrane bound, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins from breast cancer cell membrane fractions using the LTQ Orbitrap mass spectrometer. Our initial goal was to select membrane proteins from breast cancer cell lines and then to use the hydrazide method to identify the N-linked proteome as a prelude to evaluation of plasma/serum proteins from cancer patients. A combination of steps facilitated identification of the glycopeptides and also defined the glycosylation sites. In MCF-7, MDA-MB-453 and MDA-MB-468 cell membrane fractions, use of the hydrazide method facilitated an initial enrichment and site mapping of 27 N-linked glycosylation sites in 25 different proteins. However, only three N-linked glycosylated proteins, galectin-3 binding protein, lysosome associated membrane glycoprotein 1, and oxygen regulated protein, were identified in all three breast cancer cell lines. In addition, MCF-7 cells shared an additional 3 proteins with MDA-MB-453. Interestingly, the hydrazide method isolated a number of other N-linked glycoproteins also known to be involved in breast cancer, including epidermal growth factor receptor (EGFR), CD44, and the breast cancer 1, and early onset isoform 1 (BRCA1) biomarker. Analyzing the N-glycoproteins from membranes of breast cancer cell lines highlights the usefulness of the procedure for generating a practical set of potential biomarkers.

Proteomics and mass spectrometry for cancer biomarker discovery.

Proteomics is a rapidly advancing field not only in the field of biology but also in translational cancer research. In recent years, mass spectrometry and associated technologies have been explored to identify proteins or a set of proteins specific to a given disease, for the purpose of disease detection and diagnosis. Such biomarkers are being investigated in samples including cells, tissues, serum/plasma, and other types of body fluids. When sufficiently refined, proteomic technologies may pave the way for early detection of cancer or individualized therapy for cancer. Mass spectrometry approaches coupled with bioinformatic tools are being developed for biomarker discovery and validation. Understanding basic concepts and application of such technology by investigators in the field may accelerate the clinical application of protein biomarkers in disease management.

Photodynamic therapy and tumor imaging of hypericin-treated squamous cell carcinoma.

BACKGROUND: Conventional cancer therapy including surgery, radiation, and chemotherapy often are physically debilitating and largely ineffective in previously treated patients with recurrent head and neck squamous cell carcinoma (SCC). A natural photochemical, hypericin, could be a less invasive method for laser photodynamic therapy (PDT) of these recurrent head and neck malignancies. Hypericin has powerful photo-oxidizing ability, tumor localization properties, and fluorescent imaging capabilities as well as minimal dark toxicity. The current study defined hypericin PDT in vitro with human SCC cells before the cells were grown as tumor transplants in nude mice and tested as a model for hypericin induced tumor fluorescence and PDT via laser fiberoptics. METHODS: SNU squamous carcinoma cells were grown in tissue culture, detached from monolayers with trypsin, and incubated with 0.1 microg to 10 microg/ml of hypericin before exposure to laser light at 514, 550, or 593 nm to define optimal dose, time, and wavelength for PDT of tumor cells. The SCC cells also were injected subcutaneously in nude mice and grown for 6-8 weeks to form tumors before hypericin injection and insertion of fiberoptics from a KTP532 surgical laser to assess the feasibility of this operating room instrument in stimulating fluorescence and PDT of tumors. RESULTS: In vitro testing revealed a hypericin dose of 0.2-0.5 microg/ml was needed for PDT of the SCC cells with an optimal tumoricidal response seen at the 593 nm light absorption maximum. In vivo tumor retention of injected hypericin was seen for 7 to 10 days using KTP532 laser induced fluorescence and biweekly PDT via laser fiberoptics led to regression of SCC tumor transplants under 0.4 cm2 diameter, but resulted in progression of larger size tumors in the nude mice. CONCLUSION: In this preclinical study, hypericin was tested for 514-593 nm dye laser PDT of human SCC cells in vitro and for KTP532 surgical laser targeting of SCC tumors in mice. The results suggest hypericin is a potent tumor imaging agent using this surgical laser that may prove useful in defining tumor margins and possibly in sterilizing post-resection margins. Deeply penetrating pulsed infrared laser emissions will be needed for PDT of larger and more inaccessible tumors.

Hypericin and pulsed laser therapy of squamous cell cancer in vitro.

OBJECTIVE: This in vitro study compares continuous wave and pulsed laser light at longer wavelengths for activation of the phototoxic drug hypericin in human cancer cells. BACKGROUND DATA: Two-photon pulsed laser light now allows high-resolution fluorescent imaging of cancer cells and should provide deeper tissue penetration with near infrared light for improved detection as well as phototoxicity in human tumors. METHODS: Cultured Seoul National University (SNU)-1 tumor cells from a squamous cell carcinoma (SCC) were incubated with hypericin before photoirradiation at four laser wavelengths. Phototoxicity of hypericin sensitized SCC cells was measured by dimethyl thiazoldiphenyl (MTT) tetrazolium bromide cell viability assays and by confocal fluorescence microscopy via 532-nm and infrared two-photon pulsed laser light. RESULTS: Phototoxic response increased linearly with hypericin dose of 0.1-2 microM, light exposure time of 5-120 sec, and pulsed dye laser wavelengths of 514-593 nm. Light energy delivery for 50% cell phototoxicity (LD50) response was 9 joules at 514 nm, 3 joules at 550 nm, and less than 1 joule at the 593 nm hypericin light absorption maxima. Fluorescence confocal microscopy revealed membrane and perinuclear localization of hypericin in the SNU cells with membrane damage seen after excitation with visible 532 nm continuous wave light or two-photon 700-950 nm picosecond pulsed laser irradiation. CONCLUSIONS: Hypericin may be a powerful tumor targetting drug when combined with pulsed laser light in patients with recurrent head and neck SCC.

Infrared laser activation of indocyanine green inhibits growth in human pancreatic cancer.

INTRODUCTION: Indocyanine green (ICG) is a clinically-approved, water-soluble dye that generates reactive singlet oxygen when activated by infrared light. Infrared light offers the advantage of deeper tissue penetration making ICG photodynamic therapy (PDT) ideal for treatment of intra-abdominal cancers such as pancreatic adenocarcinoma. AIMS: To determine the cytotoxicity of ICG PDT in human pancreatic cancer. METHODOLOGY: MIA PaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells were incubated for 1 hour with 0 to 50 microg/mL ICG, serially washed to remove unbound dye, and then briefly exposed to infrared light from a diode laser at 0.45 W. MTT cell viability assays were performed at 72 hours post-treatment. RESULTS: Toxicity to ICG or infrared laser alone was not observed in any of the cell lines. Cell viability assays showed an ICG dose-dependent ablation when combined with laser exposure (+L). In all 3 cancer cell lines, significant growth inhibition was seen at 10 microg/mL ICG + L with nearly total ablation at 20 microg/mL ICG + L (P < 0.01). CONCLUSION: ICG PDT induces consistent and dramatic pancreatic cancer cell death. Since neither ICG nor laser alone caused toxicity, combination therapy may offer effective control of tumor growth with minimal side effects in patients with unresectable primary or metastatic pancreatic cancer.

Anticancer effect of combined intratumor cisplatin injection and interstitial KTP laser therapy on xenografted squamous cell carcinoma.

OBJECTIVE: This study was conducted to evaluate the synergistic effect of cisplatin and interstitial KTP laser therapy induced hyperthermia in vitro and in vivo (interstitial laser chemotherapy, ILCT). METHODS: In vitro study: human hypopharyngeal squamous carcinoma cell line (SNU-1041) was incubated in 1 mL of media containing various concentrations (0, 1, 2, 5, 10, 20, 50, 100 ng/mL) of cisplatin in 37 degrees and 43 degrees C for 2 hours. The viability of the cell was evaluated by MTT assay. In vivo study: human squamous cell carcinoma tumors were grown as subcutaneous transplants in nude mice and injected into tumor with cisplatin-epigel 100 microg, followed by interstitial laser therapy (ILT) via 0.6-mm diffuser fiber tips (532 nm, 1.0J/mm(3)). RESULTS: In vitro study: the viability of tumor cells incubated with 10 ng/mL of cisplatin was 62% and 28% in 37 degrees C and 43 degrees C, respectively (p = 0.02). In vivo study: the tumor volume in 4 weeks after treatment was 179 mm(3) in ILT group and 27 mm(3) in ILCT group. The ILCT group showed 8 (80%) complete remission of tumors at 4 weeks follow up, while tumor remission occurred in only 3 (30%) in ILT group (p < 0.05). CONCLUSION: These results suggest ILCT may become an effective treatment for head and neck carcinoma with minimal functional deficit.

Selective cyclooxygenase-2 inhibitor rofecoxib (Vioxx) induces expression of cell cycle arrest genes and slows tumor growth in human pancreatic cancer.

Recent studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in pancreatic adenocarcinoma and may play a critical role in this rapidly progressing form of cancer. A human pancreatic adenocarcinoma cell line, Mia PaCa-2, was incubated for 18 hours with 5 micromol/L of rofecoxib (Vioxx), a selective COX-2 inhibitor. Total RNA was isolated and gene expression analyzed by DNA microarray chips. In a separate experiment, athymic mice were orthotopically injected with 7.5 x 10(5) Mia PaCa-2 cells through a minilaparotomy. After 1 month, laparotomy was repeated to measure tumor size, and mice were randomized to receive reformulated rodent chow containing either 12.5 mg/kg/day of rofecoxib or no drug for 21 days. Tumor growth was assessed by comparing volume before and after treatment. In vitro, rofecoxib decreased gene expression of cyclin D1/PRAD1, a key component of cell cycle progression, while increasing expression of several cell cycle arrest genes, including p21/WAF1, p33/ING, GADD34, and GADD45 (P < 0.05). In vivo, tumor growth was significantly reduced in treated vs. control mice (P < 0.05). No systemic toxicity was observed in mice receiving rofecoxib. These data suggest that rofecoxib slows the growth of human pancreatic cancer through changes in gene expression that favor cell cycle arrest.

Nd:YAG laser therapy for palliation of recurrent squamous cell carcinomas in the oral cavity.

BACKGROUND AND OBJECTIVES: The objective of this study was to evaluate the outcome of laser photo-thermoablation for palliation of recurrent squamous cell tumors of the oral cavity. STUDY DESIGN/PATIENTS AND METHODS: Seventeen patients were treated with the Nd:YAG laser (power output was 50 W) delivered through a curved oral handpiece. RESULTS: Ten patients are alive, 7 with tumor remission, and 3 with persistent disease with an average follow-up of 16 months (range = 2-36). A total of 29 tumor sites received laser treatment with 17 (58%) completely ablated. Stratified by tumor site Nd:YAG treatment led to complete local response in 8/10 buccal mucosa, 2/5 retromolar trigone, 2/2 tongue, 2/5 gingiva, 1/2 floor of mouth, 2/4 hard palate. CONCLUSIONS: Nd:YAG laser treatment of recurrent oral cavity squamous cell carcinoma can be performed safely and repeated as needed to achieve tumor palliation. However, extended follow-up may be needed before convincing evidence of long-term therapeutic benefits is obtained.