A large-scale assay library for targeted protein quantification in renal cell carcinoma tissues.

Renal cell carcinoma (RCC) represents 2.2% of all cancer incidences; however, prognostic or predictive RCC biomarkers at protein level are largely missing. To support proteomics research of localized and metastatic RCC, we introduce a new library of targeted mass spectrometry assays for accurate protein quantification in malignant and normal kidney tissue. Aliquots of 86 initially localized RCC, 75 metastatic RCC and 17 adjacent non-cancerous fresh frozen tissue lysates were trypsin digested, pooled, and fractionated using hydrophilic chromatography. The fractions were analyzed using LC-MS/MS on QExactive HF-X mass spectrometer in data-dependent acquisition (DDA) mode. A resulting spectral library contains 77,817 peptides representing 7960 protein groups (FDR = 1%). Further, we confirm applicability of this library on four RCC datasets measured in data-independent acquisition (DIA) mode, demonstrating a specific quantification of a substantially increased part of RCC proteome, depending on LC-MS/MS instrumentation. Impact of sample specificity of the library on the results of targeted DIA data extraction was demonstrated by parallel analyses of two datasets by two pan human libraries. The new RCC specific library has potential to contribute to better understanding the RCC development at molecular level, leading to new diagnostic and therapeutic targets.

Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition.

The TGF-ß signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-ß signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-ß effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-ß treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-ß treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-ß have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-ß treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-ß have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

SWATH-MS Analysis of FFPE Tissues Identifies Stathmin as a Potential Marker of Endometrial Cancer in Patients Exposed to Tamoxifen.

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells.

PURPOSE: The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. METHODS: Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. RESULTS: Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. CONCLUSIONS: These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

Tamoxifen-Dependent Induction of AGR2 Is Associated with Increased Aggressiveness of Endometrial Cancer Cells.

Tamoxifen treatment in breast cancer patients is associated with increased risk of endometrial malignancies. Significantly, higher AGR2 expression was found in endometrial cancers that developed in women previously treated with tamoxifen compared to those who had not been exposed to tamoxifen. An association of elevated AGR2 level with myometrial invasion occurrence and invasion depth was also found. In vitro analyses identified a stimulatory effect of AGR2 on cellular proliferation. Although adverse tamoxifen effects on endometrial cells remain elusive, our work identifies elevated AGR2 as a candidate tamoxifen-dependent mechanism of action responsible for increased incidence of endometrial cancer.

Transgelin Contributes to a Poor Response of Metastatic Renal Cell Carcinoma to Sunitinib Treatment.

Renal cell carcinoma (RCC) represents about 2-3% of all cancers with over 400,000 new cases per year. Sunitinib, a vascular endothelial growth factor tyrosine kinase receptor inhibitor, has been used mainly for first-line treatment of metastatic clear-cell RCC with good or intermediate prognosis. However, about one-third of metastatic RCC patients do not respond to sunitinib, leading to disease progression. Here, we aim to find and characterize proteins associated with poor sunitinib response in a pilot proteomics study. Sixteen RCC tumors from patients responding (8) vs. non-responding (8) to sunitinib 3 months after treatment initiation were analyzed using data-independent acquisition mass spectrometry, together with their adjacent non-cancerous tissues. Proteomics analysis quantified 1996 protein groups (FDR = 0.01) and revealed 27 proteins deregulated between tumors non-responding vs. responding to sunitinib, representing a pattern of deregulated proteins potentially contributing to sunitinib resistance. Gene set enrichment analysis showed an up-regulation of epithelial-to-mesenchymal transition with transgelin as one of the most significantly abundant proteins. Transgelin expression was silenced by CRISPR/Cas9 and RNA interference, and the cells with reduced transgelin level exhibited significantly slower proliferation. Our data indicate that transgelin is an essential protein supporting RCC cell proliferation, which could contribute to intrinsic sunitinib resistance.

AGR2 silencing contributes to metformin-dependent sensitization of colorectal cancer cells to chemotherapy.

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells.

p63 isoforms in triple-negative breast cancer: ΔNp63 associates with the basal phenotype whereas TAp63 associates with androgen receptor, lack of BRCA mutation, PTEN and improved survival.

The TP63 gene encodes two major protein variants that differ in their N-terminal sequences and have opposing effects. In breast, ΔNp63 is expressed by immature stem/progenitor cells and mature myoepithelial/basal cells and is a characteristic feature of basal-like triple-negative breast cancers (TNBCs). The expression and potential role of TAp63 in the mammary gland and breast cancers is less clear, partly due to the lack of studies that employ p63 isoform-specific antibodies. We used immunohistochemistry with ΔNp63-specific or TAp63-specific monoclonal antibodies to investigate p63 isoforms in 236 TNBCs. TAp63, but not ΔNp63, was seen in tumour-associated lymphocytes and other stromal cells. Tumour cells showed nuclear staining for ΔNp63 in 17% of TNBCs compared to 7.3% that were positive for TAp63. Whilst most TAp63+ tumours also contained ΔNp63+ cells, the levels of the two isoforms were independent of each other. ΔNp63 associated with metaplastic and medullary cancers, and with a basal phenotype, whereas TAp63 associated with androgen receptor, BRCA1/2 wild-type status and PTEN positivity. Despite the proposed effects of p63 on proliferation, Ki67 did not correlate with either p63 isoform, nor did they associate with p53 mutation status. ΔNp63 showed no association with patient outcomes, whereas TAp63+ patients showed fewer recurrences and improved overall survival. These findings indicate that both major p63 protein isoforms are expressed in TNBCs with different tumour characteristics, indicating distinct functional activities of p63 variants in breast cancer. Analysis of individual p63 isoforms provides additional information into TNBC biology, with TAp63 expression indicating improved prognosis.

Acetylsalicylic Acid and its Potential for Chemoprevention of Colorectal Carcinoma.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAID) represent a group of medicaments inhibiting cyclooxygenase (COX) enzyme, and, in parallel, these drugs show also analgesic, antipyretic and anti-inflammatory effects. Due to their efficiency, good tolerance and easy availability, they belong to the worlds most used drugs. For decades, evidence of their anti-tumor activity has been growing, with the largest amount of published work being related to colorectal cancer (CRC). Based on both in vitro and in vivo experiments and data obtained from epidemiological and clinical studies, potential application of NSAID as chemo-preventive treatment for CRC patients is recently discussed in order to prevent development or recurrence of precanceroses and tumors. Promising treatment for such indication would be acetylsalicylic acid (ASA), which is the oldest, more than 100 years used member of the NSAID family. Nonselective irreversible COX inhibition is an important but probably not solely mechanism of its anticancer activity. Notably, wider use of ASA in chemoprevention is also prevented due to particular concerns about gastrointestinal and renal toxicity caused especially by its long-term use. AIMS: This review introduces the role of COX in tumor biology of CRC and highlights the results of the most interesting experiments illustrating the anti-tumor effect of ASA. Moreover, our work evaluates the most important published clinical analyzes of the ASA chemopreventive effect on CRC and discusses the current state. Key words: non-steroidal anti-inflammatory agents - acetylsalicylic acid - colorectal carcinoma - cyclooxygenase - chemoprevention This work was supported by the projects MEYS - NPS I - LO1413, MH CZ - DRO (MMCI, 00209805) and by Czech Science Foundation project no. 16-14829S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 10. 9. 2017.

Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients.

Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumorigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001) as well as low histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression (P<0.0001). In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients.

Influence of mutation type on prognostic and predictive values of TP53 status in primary breast cancer patients.

High rates of mutation in the TP53 tumor suppressor gene have been found in many human cancers, including breast tumors, making p53 one of the most studied proteins in oncology. However, the prognostic and predictive value of alterations in this gene remains ambiguous. To analyze the clinical value of somatic TP53 mutations, we collected clinical and molecular data on 210 women with primary breast cancer. We found significant associations of p53 mutations with tumor grade, metastasis, molecular subtype, Her2 status and inverse correlations with estrogen and progesterone receptor status. Cox proportional hazard analysis confirmed a strong prognostic value of p53 mutation for overall survival rate and highlighted significant interactions with lymph node involvement and tumor size. In relation to treatment options, TP53 mutations were associated with poor response to anthracyclines and radiotherapy. Categorization of TP53 mutations according to their type and location revealed that patients with nonsense mutation have the poorest prognosis in comparison with wild-type cases and other types of mutations in this gene. Classification of TP53 mutations with respect to the degree of disturbance of protein structure showed association of disruptive mutations with poorer patients' outcome in contrast to wild-type and non-disruptive mutations. In conclusion, the present study confirms p53 as a potential predictive and prognostic factor in oncology practice and highlights the growing evidence that distinct types of mutations have different clinical impacts.