Differentially expressed genes associated with high metabolic tumor volume served as diagnostic markers and potential therapeutic targets for pancreatic cancer.

BACKGROUND: The lack of distinct biomarkers for pancreatic cancer is a major cause of early-stage detection difficulty. The pancreatic cancer patient group with high metabolic tumor volume (MTV), one of the values measured from positron emission tomography-a confirmatory method and standard care for pancreatic cancer, showed a poorer prognosis than those with low MTV. Therefore, MTV-associated differentially expressed genes (DEGs) may be candidates for distinctive markers for pancreatic cancer. This study aimed to evaluate the possibility of MTV-related DEGs as markers or therapeutic targets for pancreatic cancer. METHODS: Tumor tissues and their normal counterparts were obtained from patients undergoing preoperative 18F-FDG PET/CT. The tissues were classified into MTV-low and MTV-high groups (7 for each) based on the MTV2.5 value of 4.5 (MTV-low: MTV2.5 < 4.5, MTV-high: MTV2.5 ≥ 4.5). Gene expression fold change was first calculated in cancer tissue compared to its normal counter and then compared between low and high MTV groups to obtain significant DEGs. To assess the suitability of the DEGs for clinical application, the correlation of the DEGs with tumor grades and clinical outcomes was analyzed in TCGA-PAAD, a large dataset without MTV information. RESULTS: Total RNA-sequencing (MTV RNA-Seq) revealed that 44 genes were upregulated and 56 were downregulated in the high MTV group. We selected the 29 genes matching MTV RNA-seq patterns in the TCGA-PAAD dataset, a large clinical dataset without MTV information, as MTV-associated genes (MAGs). In the analysis with the TCGA dataset, MAGs were significantly associated with patient survival, treatment outcomes, TCGA-PAAD-suggested markers, and CEACAM family proteins. Some MAGs showed an inverse correlation with miRNAs and were confirmed to be differentially expressed between normal and cancerous pancreatic tissues. Overexpression of KIF11 and RCC1 and underexpression of ADCY1 and SDK1 were detected in ~ 60% of grade 2 pancreatic cancer patients and associated with ~ 60% mortality in stages I and II. CONCLUSIONS: MAGs may serve as diagnostic markers and miRNA therapeutic targets for pancreatic cancer. Among the MAGs, KIF11, RCC1, ADCY, and SDK1 may be early diagnostic markers.

MicroRNA alteration and putative target genes in high-grade prostatic intraepithelial neoplasia and prostate cancer: STAT3 and ZEB1 are upregulated during prostate carcinogenesis.

BACKGROUND: We aimed to identify alteration of cancer-related miRNAs in HGPIN and PCa, and to investigate the clinical implications of HGPIN as a precancerous lesion of PCa. METHODS: Clinicopathologic analysis based on the status of HGPIN was performed in 388 patients who received radical prostatectomy between January 2005 and December 2008 in Severance Hospital. Among them, 10 paired HGPIN and PCa were prepared to perform miRNA microarray and quantitative real-time PCR. Fifty-two prostatectomy specimens were used to further validation of protein expression that was assessed by immunohistochemical staining (IHC) in matched non-neoplastic prostatic tissue (NPT), HGPIN, and PCa. Functional analysis was performed using a prostate normal cell line (RWPE-1) and two prostate cancer cell lines (LNCaP, PC-3) for comparison of expression of miR-155 and STAT3 mRNA before and after treatment of miR-155 mimetics/antagomir into each cell line. RESULTS: Patients with HGPIN had significantly less lymphovascular invasion, less lymph node metastasis, lower tumor volume, lower Gleason score, lower incidence of death, and longer overall survival compared to patients without HGPIN. MiR-155, miR-210, miR-153, and miR-200c were downregulated in HGPIN and PCa in common, compared to NPT. As putative target mRNAs, mRNA expression level of STAT3, ZEB1, and BACH1 was increased in PCa and HGPIN compared to NPT. mRNA expression level of ephrin-A3 was increased in PCa compared to NPT, and FGFRL1 was decreased in PCa compared to HGPIN and NPT. Protein expression assessed by IHC showed correlated results in STAT3, ZEB1, and ephrin-A3. Moreover, STAT3 and ZEB1 increased in a stepwise manner, from NPT to PCa. Treatment of miR-155 antagomir increased STAT3 mRNA expression in RWPE-1 cells, whereas treatment of miR-155 mimetics into PC-3 cells significantly decreased STAT3 expression. CONCLUSIONS: STAT3 and ZEB1 could be the key molecules altered at the early stages of carcinogenesis, especially in HGPIN. Prostate 76:937-947, 2016. © 2016 Wiley Periodicals, Inc.

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFß1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5ß1 and αvß3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.

CD24 induced cellular quiescence-like state and chemoresistance in ovarian cancer cells via miR-130a/301a-dependent CDK19 downregulation.

Cancer stem-like cell (CSC) is thought to be responsible for ovarian cancer recurrence. CD24 serves as a CSC marker for ovarian cancer and regulates the expression of miRNAs, which are regulators of CSC phenotypes. Therefore, CD24-regulated miRNAs may play roles in manifesting the CSC phenotypes in ovarian cancer cells. Our miRNA transcriptome analysis showed that 94 miRNAs were up or down-regulated in a CD24-high clone from an ovarian cancer patient compared to a CD24-low one. The CD24-dependent expression trend of the top 7 upregulated miRNAs (miR-199a-3p, 34c, 199a-5p, 130a, 301a, 214, 34b*) was confirmed in other 8 clones (4 clones for each group). CD24 overexpression upregulated the expression of miR-199a-3p, 34c, 199a-5p, 130a, 301a, 214, and 34b* in TOV112D (CD24-low) cells compared to the control, while CD24 knockdown downregulated the expression of miR-199a-3p, 199a-5p, 130a, 301a, and 34b* in OV90 (CD24-high) cells. miR-130a and 301a targeted CDK19, which induced a cellular quiescence-like state (increased G0/G1 phase cell population, decreased cell proliferation, decreased colony formation, and decreased RNA synthesis) and resistance to platinum-based chemotherapeutic agents. CD24 regulated the expression of miR-130a and 301a via STAT4 and YY1 phosphorylation mediated by Src and FAK. miR-130a and 301a were positively correlated in expression with CD24 in ovarian cancer patient tissues and negatively correlated with CDK19. Our results showed that CD24 expression may induce a cellular quiescence-like state and resistance to platinum-based chemotherapeutic agents in ovarian cancer via miR-130a and 301a upregulation. CD24-miR-130a/301a-CDK19 signaling axis could be a prognostic marker for or a potential therapeutic target against ovarian cancer recurrence.

CD24⁺ ovary cancer cells exhibit an invasive mesenchymal phenotype.

We recently reported that the subset of CD24(+) cells in ovarian cancer possesses various cancer stem cell properties. In this study, we further show that this subpopulation of ovarian cancer cells exhibits an epithelial-mesenchymal transition (EMT) phenotype, high invasive capacity, and CXCR4/SDF-1-mediated chemotactic migration. We evaluated CD24 expression in various ovarian cancer cell lines by flow cytometric analysis. CAOV3 and a primary ovarian cancer cell line Clone 4 were sorted into CD24(+) and CD24(-) subpopulations by FACS and Western blot, cell invasion, adhesion, and in vitro chemotaxis assays were performed with these two subpopulations. We also assessed the effects of shRNA depletion of CD24 in CAOV3 and Clone 4 cells by Western blot and cell invasion assays. CD24 expression in ovarian cancer cell lines correlated with aggressive histologic subtypes of epithelial ovarian cancer. The CD24(+) subpopulation was also more invasive than the CD24(-) subpopulation and showed higher CXCR4/SDF-1-mediated chemotactic migration. CD24(+) cells exhibited an EMT phenotype as characterized by loss of E-cadherin expression and gain of vimentin, Twist, and Snail1 expression. In addition, CD24(+) cells stimulated cell attachment to fibronectin through the activation of ß1 integrin. Depletion of CD24 expression by CD24 shRNA efficiently suppressed cell invasion and induced downregulation of CXCR4 as well as loss of the EMT phenotype. In conclusion, CD24 expression in ovarian cancer may be related to tumor aggressiveness, in particular cell invasion and chemotactic migration. Therefore, CD24 may be a good candidate for a therapeutic target for ovarian cancer.

MET overexpression in ovarian cancer via CD24-induced downregulation of miR-181a: A signalling for cellular quiescence-like state and chemoresistance in ovarian CSCs.

Increased expression of CD24 and MET, markers for cancer stem-like cells (CSCs), are each associated with ovarian cancer severity. However, whether CD24 and MET are co-expressed in ovarian CSCs and, if so, how they are related to CSC phenotype manifestation remains unknown. Our immunohistochemistry analysis showed that the co-expression of CD24 and MET was associated with poorer patient survival in ovarian cancer than those without. In addition, analyses using KM plotter and ROC plotter presented that the overexpression of CD24 or MET in ovarian cancer patients was associated with resistance to platinum-based chemotherapy. In our miRNA transcriptome and putative target genes analyses, miR-181a was downregulated in CD24-high ovarian cancer cells compared to CD24-low and predicted to bind to CD24 and MET 3'UTRs. In OV90 and SK-OV-3 cells, CD24 downregulated miR-181a expression by Src-mediated YY1 activation, leading to increased expression of MET. And, CD24 or MET knockdown or miR-181a overexpression inhibited the manifestation of CSC phenotypes, cellular quiescence-like state and chemoresistance, in OV90 and SK-OV-3 cells: increased colony formation, decreased G0/G1 phase cell population and increased sensitivity to Cisplatin and Carboplatin. Our findings suggest that CD24-miR-181a-MET may consist of a signalling route for ovarian CSCs, therefore being a combinatory set of markers and therapeutic targets for ovarian CSCs.

In situ identification and localization of IGHA2 in the breast tumor microenvironment by mass spectrometry.

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.

Invasive breast cancer induces laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling.

INTRODUCTION: Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype. METHODS: Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3ß1 or α6ß4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin ß1, or integrin ß4. RESULTS: MDA-MB-231 cells induced laminin-332 upregulation and integrin ß4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3ß1. Integrin ß4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin ß4 expression suppressed fibroblast aggregation in conditions of anoikis. CONCLUSION: Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin ß4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.

Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro.

Fibroblasts were extracted from tissue in tumor burden zones, distal normal zones and interface zones between tumor and normal tissue of human breast carcinomas, and the corresponding fibroblasts were designated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). The crosstalk between three types of fibroblasts and breast cancer cells was evaluated using an in vitro direct co-culture model. We found that INFs grew faster and expressed higher levels of fibroblast activation protein than did NFs and CAFs. Compared with CAFs and NFs, INFs grown with breast cancer cells were significantly more effective in inducing an epithelial-mesenchymal transition (EMT) in cancer cells, as indicated by induction of vimentin and N-cadherin and downregulation of E-cadherin. This EMT process was also accompanied by activation of extracellular signal-regulated kinase (ERK) and modulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Additionally, INFs promoted breast cell migration to a larger extent compared with NFs and CAFs. Taken together, these findings indicate that INFs isolated from the tumor interface zone exhibited more robust biological modulatory activity than did NFs and CAFs isolated from normal and tumor zones of the same tumor tissue, suggesting that the interface zone of the tumor represents a dynamic region vital to tumor progression.

Targeting ILK and ß4 integrin abrogates the invasive potential of ovarian cancer.

Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of ß1 and ß4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of ß1 and ß4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of ß4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of ß4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting ß4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

Laminin-332-rich tumor microenvironment for tumor invasion in the interface zone of breast cancer.

Dense fibrosis, which is caused by desmoplastic reaction, is usually found in invasive ductal carcinoma and may represent the alteration of the tumor microenvironment preceding tumor invasion. Thus, the dense fibrotic zone around invasive ductal carcinoma can be considered to be the actual tissue site of tumor microenvironment, where the precedent alterations for tumor invasion occur. To characterize the dense fibrotic zone, we classified invasive ductal carcinoma tissue into a tumor zone, a normal zone, and the novel interface zone (IZ), which shows dense fibrosis. The postulated IZ is a 5-mm-wide belt that circles the tumor margin and overlaps with normal tissue. Of the extracellular matrix components, laminin-332 was specifically overexpressed in the IZ. Events that appear to be similar to the epithelial-mesenchymal transition, a novel source of myofibroblast formation from epithelial cells, were observed in the IZ, according to the following characteristics: overexpression of matrix metalloproteinase 3, membrane type 1-matrix metalloproteinase, snail, and zinc finger E-box-binding homeobox 1, and the gain of N-cadherin expression, as well as the down-regulation of miR200c. The myofibroblasts isolated from the IZ, which were designated interface zone-fibroblast, displayed laminin-332 and membrane type 1-matrix metalloproteinase overexpression, in contrast with both cancer-associated fibroblasts and normal breast fibroblasts. Taken together, our results suggest that the IZ, which shows dense fibrosis, may provide a specialized microenvironment for guiding tumor invasion: the fibrosis caused by laminin-332 overexpressing myofibroblast formation (interface zone-fibroblast) via epithelial-mesenchymal transition.

Prospective evaluation of longitudinal changes in human papillomavirus genotype and phylogenetic clade associated with cervical disease progression.

OBJECTIVE: Changes in the HPV genotype detected in patients over time could alter cervical disease progression. Identification of patterns in the alteration of HPV genotype should also be related to cytological and histological findings. Thus, we assessed the risk for low-grade squamous intraepithelial lesion (LSIL) or high-grade SIL (HSIL)/squamous cell carcinoma (SCC) associated with alterations in the HPV genotype detected, presence of multiple HPV genotypes, and individual genotyping or HPV clade grouping. METHODS: The 1052 participants were monitored by HPV chip and Pap smear. We calculated odds ratios and applied sequential association analysis (SAA) and decision tree analysis (DTA). RESULTS: We classified HPV alteration as persistence, regression (spontaneous vs. therapeutic), or metatyping (progressive vs. regressive). Spontaneous regression occurred in 71.9% of patients. Metatyping was strongly associated with progression (RR: 3.9, p=0.0242), with progressive metatyping showing a higher risk of progression (RR: 31.49, p=0.00448). Few patients with multiple infections were identified in the initial screen but 30.8% of patients had multiple infections in the final analysis. HPV-16, -35, -52, and -58 were commonly associated with HPV persistence. Univariate analysis determined that final diagnosis significantly associated with HPV type at the endpoint (p<0.0001), persistence (p=0.0001), and progressive metatyping (p=0.0022). SAA determined that HPV-66, -68, and -69 were significantly associated with HSIL, and HPV-16 and -18 persistence significantly association with SCC. DTA indicated an age less than 28 years had a peak in LSIL, and an age between 32 and 48 years had a peak in HSIL. A bimodal peak in SCC for HR-2 at the endpoint was observed in participants less than 32 and greater than 48 years of age. CONCLUSIONS: The alteration patterns of HPV infection detected included persistence, regression, and metatyping. HPV persistence and progressive metatyping are significant signatures of disease progression.

Artificial Intelligence Techniques for Prostate Cancer Detection through Dual-Channel Tissue Feature Engineering.

The optimal diagnostic and treatment strategies for prostate cancer (PCa) are constantly changing. Given the importance of accurate diagnosis, texture analysis of stained prostate tissues is important for automatic PCa detection. We used artificial intelligence (AI) techniques to classify dual-channel tissue features extracted from Hematoxylin and Eosin (H&E) tissue images, respectively. Tissue feature engineering was performed to extract first-order statistic (FOS)-based textural features from each stained channel, and cancer classification between benign and malignant was carried out based on important features. Recursive feature elimination (RFE) and one-way analysis of variance (ANOVA) methods were used to identify significant features, which provided the best five features out of the extracted six features. The AI techniques used in this study for binary classification (benign vs. malignant and low-grade vs. high-grade) were support vector machine (SVM), logistic regression (LR), bagging tree, boosting tree, and dual-channel bidirectional long short-term memory (DC-BiLSTM) network. Further, a comparative analysis was carried out between the AI algorithms. Two different datasets were used for PCa classification. Out of these, the first dataset (private) was used for training and testing the AI models and the second dataset (public) was used only for testing to evaluate model performance. The automatic AI classification system performed well and showed satisfactory results according to the hypothesis of this study.

Molecular proteomics imaging of tumor interfaces by mass spectrometry.

The specific molecular profiles of ovarian cancer interface zones (IZ), the region between tumors and normal tissues, were evaluated using a new method involving matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS). We analyzed three ovarian serous carcinomas using MALDI-IMS. Principal component analysis (PCA) was used to evaluate the quality of tissue spatial features based on MALDI-IMS, and for analysis of large data sets of MALDI-IMS. Two-dimensional gel electrophoresis and fluorescence microscopy were used to verify interface-specific proteins. Unique profiles were identified for the tumors, the normal zone, and the IZ. Through MALDI analysis, two interface-specific proteins, plastin 2 and peroxiredoxin 1 (PRDX 1), were identified as differentially regulated between zones. Fluorescence microscopy revealed high expression levels of plastin 2 and PRDX 1 along the IZ of ovarian tumors. This comparative proteomics study using tissue MALDI-IMS suggested that the IZ is different from the adjacent tumor and normal zones, and that plastin 2 and PRDX 1 may be interface markers specific to ovarian tumors.

Proteomic molecular portrait of interface zone in breast cancer.

Surgical tumor margins are intended to encompass residual tumor cells but may not always accurately delineate the boundary between tumor and normal tissue. Efforts to define tumor margins based on molecular analysis have achieved limited success. Furthermore, no clinical trials have addressed the scope of the tumor microenvironment. Here, we considered the tumor cell population and surrounding microenvironment in delineating tumor margins, classifying breast cancer into tumor and normal zones, and introducing the concept of an interface zone, the region between the invading tumor front and normal tissue, which develops during tumor invasion and metastasis through remodeling of the tumor microenvironment. Pathological signatures of invasion markers in tumor tissues are most dynamic within the invading tumor front. We compared protein profiles of tumor, normal, and interface zones using MALDI-MS. Proteins upregulated in the interface zone were identified by peptide mass fingerprinting and confirmed by database searching with chemically assisted MALDI-PSD spectra. Upregulation was confirmed for RhoGDIα, CAPG, WDR1, and CK8 by Western and immunohistochemical analyses. Our results demonstrate that the molecular profile of the interface zone is unique and suggest that upregulation of proteins here may be related to progression and metastasis of breast carcinomas.

Panel of candidate biomarkers for renal cell carcinoma.

The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by the lack of specific biomarkers. To identify candidate RCC biomarkers, we used 2-DE gel electrophoresis with mass spectrometry and 2-DE spot intensity-based ROC analysis to analyze 18 sets of paired normal and RCC tumor tissue including conventional, papillary, and chromophobe subtypes. Validation was performed with RCC patient plasma samples and confirmed by clustergram, shRNA, and immunohistochemistry assays. Cardinal candidates were evaluated by ELISA. The leading candidate biomarker that was upregulated in RCC samples according to the clustergram and validation analysis was nicotinamide N-methyltransferase (NNMT) (13/15, P < 0.0001). Other upregulated candidate biomarkers that were identified by this method include ferritin, hNSE, NM23, secretagogin, and L-plastin. The upregulation of NNMT in RCC was confirmed by immunoblotting and immunohistochemistry. Analysis of fractionated membrane-associated proteins identified CAP-G, mitofillin, tubulin alpha, RBBP7, and HSP27. Of these, RBBP7 and HSP27 were highly expressed in the chromophobe subtype of RCC (3/3) but were absent from conventional RCC (0/3). The triple combination of the NNMT, FTL, and hNSE biomarkers had the highest predictive capacity of 0.993, while NNMT was the single, most powerful candidate diagnostic biomarker for all types of RCC.

ITGB4-mediated metabolic reprogramming of cancer-associated fibroblasts.

Integrin beta 4 (ITGB4) overexpression in cancer cells contributes to cancer progression. However, the role of stromal ITGB4 expression in cancer progression remains poorly understood, despite stromal ITGB4 overexpression in malignant cancers. In our study, ITGB4-overexpressing triple negative breast cancer (TNBC) cells provided cancer-associated fibroblasts (CAFs) with ITGB4 proteins via exosomes, which induced BNIP3L-dependent mitophagy and lactate production in CAFs. In coculture assays, the ITGB4-induced mitophagy and glycolysis were suppressed in CAFs by knocking down ITGB4 or inhibiting exosome generation in MDA-MB-231, or blocking c-Jun or AMPK phosphorylation in CAFs. ITGB4-overexpressing CAF-conditioned medium promoted the proliferation, epithelial-to-mesenchymal transition, and invasion of breast cancer cells. In a co-transplant mouse model, MDA-MB-231 made a bigger tumor mass with CAFs than ITGB4 knockdown MDA-MB-231. Herein, we presented how TNBC-derived ITGB4 protein triggers glycolysis in CAFs via BNIP3L-dependent mitophagy and suggested the possibility that ITGB4-induced mitophagy could be targeted as a cancer therapy.

The stromal loss of miR-4516 promotes the FOSL1-dependent proliferation and malignancy of triple negative breast cancer.

Stroma-derived exosomal microRNA (exomiR) contributes to tumor progression, however, which remains poorly understood. In our study, we analyzed exomiRs from the cancer-associated fibroblast (CAF) and normal fibroblast (NF) isolated from an invasive ductal carcinoma (IDC) patient and found that the level of microRNA (miR)-4516 was approximately 5-fold lower in CAF-derived exosomes than NF-derived ones. In gene annotation analysis, miR-4516 target genes were mainly associated with the regulation of proliferation. miR-4516 overexpression or mimic treatment suppressed the proliferation of breast cancer cells, especially triple negative breast cancer (TNBC) cells. Among miR-4516 targets, FOSL1 was overexpressed in TNBC cells compared to non-TNBC cells and promoted tumor proliferation. The expression of miR-4516 and FOSL1 was reversely correlated in breast cancer patient tissues. Particularly, TNBC patients with high FOSL1 expression showed a significant poorer survival than those with low FOSL1 expression. Our results show that the loss of miR-4516 from CAF-derived exosomes is associated with FOSL1-dependent TNBC progression and suggest that miR-4516 can be used as an anti-cancer drug for TNBC.

Scale-Up Evaluation of a Composite Tumor Marker Assay for the Early Detection of Renal Cell Carcinoma.

The early detection of renal cell carcinoma (RCC) using tumor markers remains an attractive prospect for the potential to downstage the disease. To validate the scale-up clinical performance of potential tumor markers for RCC (as a single marker and as a composite tumor marker composed of nicotinamide N-methyltransferase (NNMT), L-Plastin (LCP1), and non-metastatic cells 1 protein (NM23A)), the scale-up assay was performed. Patients with RCC from multiple domestic institutes were included in the clinical evaluation for reassessment and improvement of the established triple markers of our product. For the diagnostic performance of the composite markers, the best-split cutoff points of each marker (147 pg/mL for NNMT, 1780 pg/mL for LCP1, and 520 pg/mL for NM23A) were installed. Serum levels of NNMT, LCP1, and NM23A were greatly increased in subjects with RCC (p < 0.0001). In 1042 blind sample tests with control individuals (n = 500) and patients with RCC (n = 542), the diagnostic sensitivity and specificity of the composite three-marker assay were 0.871 and 0.894, respectively, and the resulting AUC (Area under Curve) of ROC (Receiver Operating Characteristic) was 0.917. As a single marker, the diagnostic accuracies of NNMT, LCP1, and NM23A, as estimated by ROC, were 0.833, 0.844, and 0.601, respectively. The composite three-marker assay with NNMT, LCP1, and NM23A is a more improved novel serum marker assay for the early detection of RCC in cases of renal mass or unknown condition. The NNMT, LCP1, and NM23A triple marker assay could be a powerful diagnostic tumor marker assay to screen the early stage of RCC.