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.

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.

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.

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.

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.

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.

Compression-induced expression of glycolysis genes in CAFs correlates with EMT and angiogenesis gene expression in breast cancer.

Tumor growth increases compressive stress within a tissue, which is associated with solid tumor progression. However, very little is known about how compressive stress contributes to tumor progression. Here, we show that compressive stress induces glycolysis in human breast cancer associated fibroblast (CAF) cells and thereby contributes to the expression of epithelial to mesenchymal (EMT)- and angiogenesis-related genes in breast cancer cells. Lactate production was increased in compressed CAF cells, in a manner dependent on the expression of metabolic genes ENO2, HK2, and PFKFB3. Conditioned medium from compressed CAFs promoted the proliferation of breast cancer cells and the expression of EMT and/or angiogenesis-related genes. In patient tissues with high compressive stress, the expression of compression-induced metabolic genes was significantly and positively correlated with EMT and/or angiogenesis-related gene expression and metastasis size. These findings illustrate a mechanotransduction pathway involving stromal glycolysis that may be relevant also for other solid tumours.

HSP27, ALDH6A1 and Prohibitin Act as a Trio-biomarker to Predict Survival in Late Metastatic Prostate Cancer.

BACKGROUND/AIM: The aim of this study was to evaluate the usefulness of biomarkers related to prostate cancer metastasis and survival of patients. MATERIALS AND METHODS: Proteomics were used for detecting significant differences in protein expression among normal prostate, localized prostate cancer and metastatic cancer using 2-dimensional gel electrophoresis and mass spectrometry. mRNA expression was then examined in order to further confirm significant differences in protein expression. A total of 7 proteins were found to be differentially expressed. Immunochemistry (IHC), was also used to confirm the levels of expression of the 7 proteins in prostate cancer. Survival analysis using the candidate markers was finally performed in 98 metastatic prostate cancer patients according to IHC results. RESULTS: In metastatic lesions, proteomic analysis indicated that heat shock protein (HSP) 27, prohibitin, glutathione S-transferase 1, fibrinogen ß chain, and aldehyde dehydrogenase 6A1 were up-regulated, while α1 antitrypsin, and HSP 60 were down-regulated. IHC revealed that HSP 27, ALDH6A1 and prohibitin were highly specific to metastatic tumor cells. HSP27 and prohibitin appeared more strongly in the incipient stage of cancer than metastatic cancer, and ALDH6A1 was significantly reduced in metastatic cancer (p<0.01). Of all proteins, phohibitin had the highest value in predicting survival. However, all three proteins were a stronger marker than each one separately. CONCLUSION: Trio-biomarker composed of HSP27, ALDH6A1 and prohibitin may predict survival of metastatic prostate cancer patients.

Nodal metastasis signatures in breast cancer.

Although the molecular taxonomy of invasive breast cancer is based on heterogeneous histologic types, pathologic nodal (pN) stage remains one of the most important independent prognostic factors. Although node-positive number (NPN) has been widely as an accepted staging algorithm of pN stage, the node-positive ratio (NPR) in totally resected axillary nodes has been considered as another reasonable indicator. We aimed to identify signatures to play a predictive role in nodal metastasis for analytic validation between the primary breast cancers with positive node metastasis and those with negative node metastasis. We validated expression profiles of surrogate candidates extracted from the prior 2D MALDI-TOF data for invasive breast cancer using fluorescence/silver in situ hybridization (FISH/SISH) and immunohistochemistry (IHC) in 151 primary breast cancers accompanied with 102 metastatic nodal tissues. Cox proportional hazards regression analyses indicated that event factors (recurrence or metastasis) were significantly more frequent in cases with CCDN1, c-myc gene amplification, IgHA2 low expression. CCDN1 gene amplification (OR: 5.702, p=0.0006), IgHA2 low expression (OR: 0.16, p=0.0184) remained significant factors for events on multivariate analyses. WDR+/ERK++ was significantly detected in higher pN stage (averaging 6.5 regional nodes or 43% of NPR), while seldom found in pN0-1. In conclusion, both overexpression of WDR1 and p-ERK in the primary breast cancer could play a role in the nodal signature over pN2-3.

Mechanical compression induces VEGFA overexpression in breast cancer via DNMT3A-dependent miR-9 downregulation.

Tumor growth generates mechanical compression, which may trigger mechanotransduction in cancer and stromal cells and promote tumor progression. However, very little is known about how compression stimulates signal transduction and contributes to tumor progression. In the present study, we demonstrated that compression enhances a tumor progression phenotype using an in vitro compression model, and validated the results from the in vitro model with high- and low-compressed breast cancer tissues. Mechanical compression induced miR-9 downregulation by DNMT3A-dependent promoter methylation in the MDA-MB-231 and BT-474 breast cancer cell lines and in cancer-associated fibroblasts. The overexpression of miR-9 target genes (LAMC2, ITGA6, and EIF4E) was induced by miR-9 downregulation, which eventually enhanced vascular endothelial growth factors production. Demethylation and decompression could reverse compression-induced miR-9 downregulation and following overexpression of miR-9 target genes and VEGFA.

Ovarian Clear Cell Carcinoma Sub-Typing by ARID1A Expression.

PURPOSE: Loss of AT-rich DNA-interacting domain 1A (ARID1A) has been identified as a driving mutation of ovarian clear cell carcinoma (O-CCC), a triple-negative ovarian cancer that is intermediary between serous and endometrioid subtypes, in regards to molecular and clinical behaviors. However, about half of O-CCCs still express BAF250a, the protein encoded by ARID1A. Herein, we aimed to identify signatures of ARID1A-positive O-CCC in comparison with its ARID1A-negative counterpart. MATERIALS AND METHODS: Seventy cases of O-CCC were included in this study. Histologic grades and patterns of primary tumor, molecular marker immunohistochemistry profiles, and clinical outcomes were analyzed. RESULTS: Forty-eight (69%) O-CCCs did not express BAF250a, which were designated as "ARID1A-negative." The other 22 (31%) O-CCCs were designated as "ARID1A-positive." ARID1A-positive tumors were more likely to be histologically of high grades (41% vs. 10%, p=0.003), ERß-positive (45% vs. 17%, p=0.011), and less likely to be HNF1ß-positive (77% vs. 96%, p=0.016) and E-cadherin-positive (59% vs. 83%, p=0.028) than ARID1A-negative tumors. Patient age, parity, tumor stage were not significantly different in between the two groups. Cancer-specific survival was not significantly different either. CONCLUSION: We classified O-CCCs according to ARID1A expression status. ARID1A-positive O-CCCs exhibited distinct immunohistochemical features from ARID1A-negative tumors, suggesting a different underlying molecular event during carcinogenesis.

STAT3-induced WDR1 overexpression promotes breast cancer cell migration.

WD repeat domain 1 (WDR1), a protein that assists cofilin-mediated actin filament disassembly, is overexpressed in the invading front of invasive ductal carcinoma (IDC), but its implication of overexpression and how to be regulated have not been studied. In our study, we demonstrated that STAT3 bound to the 5' upstream sequence (-1971 to -1964), a putative promoter region, of WDR1 gene, and its activation induced WDR1 overexpression in breast cancer cells. The exogenous overexpression of WDR1 increased the migration of MDA-MB-231, which was attenuated by WDR1 knockdown. In the analysis of breast cancer patients, WDR1 overexpression was associated with a shorter distant metastasis-free survival (DMFS), more specifically in basal-like tumors.

Angiopoietin-2 promotes ER+ breast cancer cell survival in bone marrow niche.

In estrogen receptor-positive (ER+) breast cancer, it is recognized that metastases may develop after a long period of dormancy. Bone marrow (BM) vascular niche is where the dormant tumor cells are most likely to reside. So far, it is not fully understood why the dormant tumor cells become proliferative and eventually generate tumor. We hypothesized that therapeutic or menopause-related estrogen depletion may be the switch behind dormant ER+ tumor cell awakening in BM. We utilized an existing experimental model of BM endothelial niche that can simulate ER+ tumor cell dormancy to test our hypothesis. In results, estrogen depletion paradoxically promoted ER+ tumor cell proliferation in the BM endothelial niche, and their molecular phenotype shifted from dormant to awaken. Following estrogen depletion, the BM niche cells produced angiopoietin-2 (ANGPT2), which destabilized niche endothelium by interfering ANGPT1/Tie2 signaling, and promoted ER+ tumor cell survival under estrogen deficiency via cell surface integrin &1. Knockdown of ANGPT2 completely negated ER+ tumor cell awakening in the niche. Furthermore, ANGPT2 expression in ER+ tumor human samples was associated with increased risk of distant metastasis only in those who underwent adjuvant estrogen depletion therapy, not in those who did not undergo adjuvant therapy. In conclusion, we demonstrate that ANGPT2 signaling activated after estrogen depletion paradoxically triggers ER+ tumor cell awakening from dormancy in their BM niche, partly indirectly via endothelial Tie2 receptor and partly directly via tumor cell surface integrin &1.

Transcriptome-wide analysis of compression-induced microRNA expression alteration in breast cancer for mining therapeutic targets.

Tumor growth-generated mechanical compression may increase or decrease expression of microRNAs, leading to tumor progression. However, little is known about whether mechanical compression induces aberrant expression of microRNAs in cancer and stromal cells. To investigate the relationship between compression and microRNA expression, microRNA array analysis was performed with breast cancer cell lines and cancer-associated fibroblasts (CAFs) exposed to different compressive conditions. In our study, mechanical compression induced alteration of microRNA expression level in breast cancer cells and CAFs. The alteration was greater in the breast cancer cells than CAFs. Mechanical compression mainly induced upregulation of microRNAs rather than downregulation. In a parallel mRNA array analysis, more than 25% of downregulated target genes were functionally involved in tumor suppression (apoptosis, cell adhesion, and cell cycle arrest), whereas generally less than 15% were associated with tumor progression (epithelial-mesenchymal transition, migration, invasion, and angiogenesis). Of all cells examined, MDA-MB-231 cells showed the largest number of compression-upregulated microRNAs. miR-4769-5p and miR-4446-3p were upregulated by compression in both MDA-MB-231 cells and CAFs. Our results suggest that mechanical compression induces changes in microRNA expression level, which contribute to tumor progression. In addition, miR-4769-5p and miR-4446-3p may be potential therapeutic targets for incurable cancers, such as triple negative breast cancer, in that this would reduce or prevent downregulation of tumor-suppressing genes in both the tumor and its microenvironment simultaneously.

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.

Estrogen Receptor Status Predicts Late-Onset Skeletal Recurrence in Breast Cancer Patients.

Estrogen receptor-positive (ER+) breast cancer (BCa) often recurs after long latency, and is known to favor bone as a metastatic site. We hypothesized that skeletal recurrence of ER+ BCa follows a different chronological pattern from that of nonskeletal recurrence.We retrospectively evaluated 434 matched pairs of ER+ and ER- female patients who underwent surgery for clinically localized BCa between 2005 and 2009. Patient age, tumor size, lymph node involvement, and adjuvant treatment biases were adjusted by the propensity score method. We conducted competing risk analysis to determine the prognostic significance of ER expression status on the risk of overall recurrence and late recurrence (after 3 years). We also compared chronological patterns of ER+ and ER- tumor recurrence, stratified by the first metastatic site (skeletal vs nonskeletal).After 3 postoperative years, ER+ tumor had a significantly higher risk of overall distant recurrence than ER- tumor (P = 0.02). When further stratified by first site of metastasis, only late skeletal recurrence was significantly associated with ER status (P = 0.029). In multivariate analysis, ER and lymph node involvement status were significant prognostic factors for late skeletal recurrence, with adjusted hazard ratios of 5.2 (95% CI = 1.2-22.4, P = 0.025) and 5.2 (1.7-16.3, P = 0.005), respectively. For nonskeletal distant recurrence, tumor size (>2 cm) was the only significant risk factor with adjusted hazard ratio of 2.8 (1.4-5.7, P = 0.005). Annual hazard of skeletal recurrence events of ER+ tumors continued to exist up to 10 years, while annual hazard of nonskeletal recurrences decreased after peaking at 5 years. ER- tumor recurrences exhibited similar annual hazard patterns across skeletal and nonskeletal sites.ER expression and lymph node involvement status were strong predictors of BCa late-onset (>3 years) recurrences, especially in skeletal sites. Therefore, skeletal system surveillance is mandatory for long-term follow-up of this subpopulation.

Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.

Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA.

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.