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BACKGROUND & AIMSPrimary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multi-focal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear and therapeutic options for PSC patients are limited. We performed cell-free messenger RNA (cf-mRNA) next generation sequencing (RNA-Seq) to characterize the circulating transcriptome of PSC and non-invasively investigate potentially bioactive signals that are associated with PSC.METHODSSerum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with non-alcoholic fatty liver disease (NAFLD). Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were built using PSC dysregulated cf-mRNA genes.RESULTSDifferential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and the liver diseases (NAFL and Non-Alcohol Steatohepatitis (NASH)) or healthy controls shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, hepatic stellate cells and Kupffer cells, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA classifier using liver-specific genes which discriminated PSC from healthy control subjects using gene transcripts of liver origin.CONCLUSIONSBlood-based whole transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in PSC subject sera which may be used to diagnose PSC patients. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for non-invasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.Graphical Abstract

Supplementary Figure-5. Curcumin and AKBA modulate genes involved in cell cycle regulation.

Supplementary Figure-1. Inhibitory concentration 50 of curcumin and AKBA.

Supplementary Figure-8. Comparative gene-expression analysis of FBXW7, cMyc, CDK4, CDK6, Cyclin E1 and Cyclin D1 from xenograft tumors assessed by qRT-PCR.

Colorectal cancer is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are well-established dietary botanicals with potent antitumorigenic properties that have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may, in part, be mediated through regulation of key cancer-related microRNAs (miRNA) and their downstream gene targets. Here, we investigated the antitumorigenic effects of curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in colorectal cancer cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell-cycle arrest in colorectal cancer cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways, including key cell-cycle regulatory genes. Combined bioinformatics and in silico analysis identified apoptosis, proliferation, and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anticancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in colorectal cancer cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein, we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer. Cancer Prev Res; 8(5); 431–43. ©2015 AACR.

Supplementary methods and tables

Supplementary Figure-7. The effects of miR-27a knockdown on cell function in colorectal cancer cells.

Supplementary Figure-4. The effects of curcumin and AKBA on miR-34a, miR-27a, miR-16 and miR-145 expression in CRC cell lines.

SYBR green qRT-PCR Primers .

Supplementary Figure-2. Curcumin and AKBA exert cytotoxicity and suppress colony formation on CRC cell lines.

Supplementary Figure-3. Curcumin and AKBA inhibit cellular proliferation in 3D cultures.

Supplementary tables and Figure Legends

Supplementary Figure-6. The effects of miR-34a over-expression on cell function colorectal cancer cells.

Although non-coding RNAs have long been considered as non-functional “junk” RNAs, accumulating evidence in the past decade indicates that they play a critical role in pathogenesis of various cancers. In addition to their biological significance, the recognition that their expression levels are frequently dysregulated in multiple cancers have fueled the interest for exploiting their clinical potential as cancer biomarkers. In particular, microRNAs (miRNAs), a subclass of small non-coding RNAs that epigenetically modulate gene-transcription, have become one of the most well-studied substrates for the development of liquid biopsy biomarkers for cancer patients. The emergence of high-throughput sequencing technologies has enabled comprehensive molecular characterisation of various non-coding RNA expression profiles in multiple cancers. Furthermore, technological advances for quantifying lowly expressed RNAs in the circulation have facilitated robust identification of previously unrecognised and undetectable biomarkers in cancer patients. Here we summarise the latest progress on the utilisation of non-coding RNAs as non-invasive cancer biomarkers. We evaluated the suitability of multiple non-coding RNA types as blood-based cancer biomarkers and examined the impact of recent technological breakthroughs on the development of non-invasive molecular biomarkers in cancer.

Supplementary tables

Supplementary figures 1-5

Cancer has emerged as a leading cause of mortality worldwide, claiming more than 8 million lives annually. Gastrointestinal cancers account for about 35% of these mortalities. Recent advances in diagnostic and treatment strategies have reduced mortality among patients with gastrointestinal cancer, yet a significant number of patients still develop late-stage cancer, where treatment options are inadequate. Emerging interests in “liquid biopsies” have encouraged investigators to identify and develop clinically relevant noninvasive genomic and epigenomic signatures that can be exploited as biomarkers capable of detecting premalignant and early-stage cancers. In this context, microRNAs (miRNA), which are small, noncoding RNAs that are frequently dysregulated in cancers, have emerged as promising entities for such diagnostic purposes. Even though the future looks promising, current approaches for detecting miRNAs in blood and other biofluids remain inadequate. This review summarizes existing efforts to exploit circulating miRNAs as cancer biomarkers and evaluates their potential and challenges as liquid biopsy–based biomarkers for gastrointestinal cancers. Clin Cancer Res; 23(10); 2391–9. ©2017 AACR.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients.Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings.Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes.Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.

Purpose: Dysregulated expression of miRNAs has emerged as a hallmark feature in human cancers. Exportin-5 (XPO5), a karyopherin family member, is a key protein responsible for transporting precursor miRNAs from the nucleus to the cytoplasm. Although XPO5 is one of the key regulators of miRNA biogenesis, its functional role and potential clinical significance in colorectal cancer remains unclear.Experimental Design: The expression levels of XPO5 were initially assessed in three genomic datasets, followed by determination and validation of the relationship between XPO5 expression and clinicopathologic features in two independent colorectal cancer patient cohorts. A functional characterization of XPO5 in colorectal cancer was examined by targeted gene silencing in colorectal cancer cell lines and a xenograft animal model.Results: XPO5 is upregulated, both at mRNA and protein levels, in colorectal cancers compared with normal tissues. High XPO5 expression is associated with worse clinicopathologic features and poor survival in colorectal cancer patient cohorts. The siRNA knockdown of XPO5 resulted in reduced cellular proliferation, attenuated invasion, induction of G1–S cell-cycle arrest, and downregulation of key oncogenic miRNAs in colorectal cancer cells. These findings were confirmed in a xenograft animal model, wherein silencing of XPO5 resulted in the attenuation of tumor growth.Conclusions: XPO5 acts like an oncogene in colorectal cancer by regulating the expression of miRNAs and may be a potential therapeutic target in colorectal cancer. Clin Cancer Res; 23(5); 1312–22. ©2016 AACR.

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear.Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model.Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947–57. ©2016 AACR.

Supplementary Tables S1 and S2

Supplementary figures and Table legends

Supplementary Table 1: Clinicopathological features of patients according to XPO5 expression (original validation cohort-2) Supplementary Table 2: Multivariate analysis for the predictors of disease-free survival in patients with colorectal cancer (original validation cohort-2) Supplementary Fig. 1: XPO5 expression analysis in discovery cohort. Supplementary Fig. 2: Clinicopathological significance of XPO5 expression in CRC patients Supplementary Fig. 3: The relationship between microsatellite status and XPO5 expression Supplementary Fig. 4: Intracellular localization of XPO5 Supplementary Fig. 5: pri-miRNA expression in siXPO5 CRC cells

Supplementary methods and figure legends

Supplementary Figure 1: The siRNA knockdown efficiency of FOXM1 and FOXQ1 in CRC cells; Supplementary Figure-2: Down-regulation of FOXM1 and FOXQ1 inhibited migration in CRC cell lines; Supplementary Figure-3:Down-regulation of FOXM1 and FOXQ1 inhibited invasion in CRC cell lines; Supplementary Figure-4: MiR-342 directly targets 3'-UTR of FOXM1 and FOXQ1 in HT-29 CRC cells; Supplementary Figure-5: MiR-342 overexpression inhibited cell migration and invasion in SW480 CRC cells; Supplementary Figure 6: Inhibition of miR-342 promotes cell migration and invasion in SW480 CRC cells; Supplementary Figure 7: MiR-342 inhibited cell migration and invasion in HT-29 CRC cells; Supplementary Figure-8: MiR-342 overexpression resulted in down-regulation of FOXM1 and FOXQ1 expression in xenograft tissues.

While best known for its role in oxidative DNA damage repair, apurinic-apyrimidinic endonuclease-1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression.

Accumulating evidence suggests that dysregulation of transcriptional enhancers plays a significant role in cancer pathogenesis. Herein, we performed a genome-wide discovery of enhancer elements in colorectal cancer (CRC). We identified PVT1 locus as a previously unrecognized transcriptional regulator in CRC with a significantly high enhancer activity, which ultimately was responsible for regulating the expression of MYC oncogene. High expression of the PVT1 long-non-coding RNA (lncRNA) transcribed from the PVT1 locus was associated with poor survival among patients with stage II and III CRCs (p PVT1 locus inversely correlated with the reduced expression of the corresponding the PVT1 lncRNA, as well as MYC gene expression. Bioinformatic analyses of CRC-transcriptomes revealed that the PVT1 locus may also broadly impact the expression and function of other key genes within two key CRC-associated signaling pathways – the TGFβ/SMAD and Wnt/β-Catenin pathways. We conclude that the PVT1 is a novel oncogenic enhancer of MYC and its activity is controlled through epigenetic regulation mediated through aberrant methylation in CRC. Our findings also suggest that the PVT1 lncRNA expression is a promising prognostic biomarker and a potential therapeutic target in CRC.

Accumulating evidence suggests that aspirin has anti-tumorigenic properties in colorectal cancer (CRC). Herein, we undertook a comprehensive and systematic series of in vivo animal experiments followed by 3D-mathematical modeling to determine the kinetics of aspirin’s anti-cancer effects on CRC growth. In this study, CRC xenografts were generated using four CRC cell lines with and without PIK3CA mutations and microsatellite instability, and the animals were administered with various aspirin doses (0, 15, 50, and 100 mg/kg) for 2 weeks. Cell proliferation, apoptosis and protein expression were evaluated, followed by 3D-mathematical modeling analysis to estimate cellular division and death rates and their impact on aspirin-mediated changes on tumor growth. We observed that aspirin resulted in a dose-dependent decrease in the cell division rate, and a concomitant increase in the cell death rates in xenografts from all cell lines. Aspirin significantly inhibited cell proliferation as measured by Ki67 staining (P < 0.05–0.01). The negative effect of aspirin on the rate of tumor cell proliferation was more significant in xenograft tumors derived from PIK3CA mutant versus wild-type cells. A computational model of 3D-tumor growth suggests that the growth inhibitory effect of aspirin on the tumor growth kinetics is due to a reduction of tumor colony formation, and that this effect is sufficiently strong to be an important contributor to the reduction of CRC incidence in aspirin-treated patients. In conclusion, we provide a detailed kinetics of aspirin-mediated inhibition of tumor cell proliferation, which support the epidemiological data for the observed protective effect of aspirin in CRC patients.

Inflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signaling pathways of innate and adaptive immune systems. Therefore, there is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses. Here we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling to assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples. Considering that, both organspecific recruitment of immune cells and organ resident bespoke immune cells contributes to restoration of organ homeostasis, we also examined LPS-induced gene-expression dysregulation of multiple organs to shed light on organ crosstalk. Cf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. Lastly, we identified the dysregulation of liverspecific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this biological compartment. Collectively, using a preclinical model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses.Abstract Figure

Key Points Question Can circulating microRNAs be used to derive clinically significant noninvasive diagnostic biomarkers for gastric cancer? Findings This diagnostic study used a multistep and comprehensive biomarker discovery approach to establish a novel, noninvasive, microRNA-based signature for the early detection of gastric cancer, which was retrospectively and prospectively validated in multicenter patient cohorts. Meaning For patients with gastric cancer and individuals with a high risk for gastric cancer, a microRNA-based signature may improve the early detection of gastric cancer., This diagnostic study describes the development and validation of a microRNA-based liquid biopsy assay for early detection of gastric cancer., Importance Noninvasive detection of early-stage disease is a key strategy for reducing gastric cancer (GC)-associated patient mortality. Objective To establish a novel, noninvasive, microRNA (miRNA)-based signature for the early detection of GC using a comprehensive biomarker discovery approach with retrospective and prospective validation. Design, Setting, and Participants This diagnostic study was conducted in 4 phases using publicly available genome sequences and tissue samples from patients at an academic medical center in Japan, and validated with retrospective multicenter cohorts of patients with GC. Three tissue miRNA data sets were used to identify a miRNA signature that discriminated GC vs normal tissues. The robustness of this signature was assessed in serum from 2 retrospective cohorts of patients with GC. A risk-scoring model was derived, then the performance of the miRNA signature was evaluated in a prospective cohort of patients with GC. The robustness of the miRNA signature was compared with current blood-based markers, and a cost-effectiveness analysis of the miRNA signature against the current practice of endoscopy was performed. All clinical samples used for this study were collected and data analyzed between April 1997 and March 2018. Main Outcomes and Measures Assessment of diagnostic efficiency on the basis of area under the curve (AUC), specificity, and sensitivity. Results The data sets for the genome-wide expression profiling analysis stage included 598 total patient samples (284 [55.4%] from men; mean [SE] patient age, 65.7 [0.5] years). The resulting 10-miRNA signature was validated in 2 retrospective GC serum cohorts (586 patients; 348 [59.4%] men, mean [SE] age, 66.0 [0.7] years), which led to the establishment of a 5-miRNA signature (AUC, 0.90; 95% CI, 0.85-0.94) that also exhibited high levels of diagnostic performance in patients with stage I disease (AUC, 0.89; 95% CI, 0.83-0.94). A risk-scoring model was derived and the assay was optimized to a minimal number of miRNAs. The performance of the resulting 3-miRNA signature was then validated in a prospective cohort of patients with GC (349 patients; 124 [70.5%] men, median [range] age, 66.0 [0.66] years). The final 3-miRNA signature (miR-18a, miR-181b, and miR-335) exhibited high diagnostic accuracy in all stages of patients (AUC, 0.86; 95% CI 0.83-0.90), including in patients with stage I disease (AUC, 0.85; 95% CI, 0.79-0.91). Furthermore, this miRNA signature was superior to currently used blood markers and outperformed the endoscopic screening in a cost-effectiveness analysis (incremental cost-effectiveness ratio, CNY ¥16162.5 per quality-adjusted life-year [USD $2304.80 per quality-adjusted life-year]). Conclusions and Relevance These results suggest the potential clinical significance of the 3-miRNA signature as a noninvasive, cost-effective, and facile assay for the early detection of GC.

OBJECTIVE: This study aimed to do a genomewide transcriptomic profiling to develop a miRNA-based signature for the identification of peritoneal metastasis (PM) in patients with gastric cancer (GC). SUMMARY BACKGROUND DATA: Even though PM in patients with GC has long been recognized to associate with poor survival, currently there is lack of availability of molecular biomarkers for its robust diagnosis. METHODS: We performed a systematic biomarker discovery by analyzing miRNA expression profiles in primary tumors from GC patients with and without PM, and subsequently validated the expression of candidate miRNA biomarkers in three independent clinical cohorts of 354 patients with advanced GC. RESULTS: Five miRNAs (miR-30a-5p, -134-5p, -337-3p, -659-3p, and -3917) were identified during the initial discovery phase; three of which (miR-30a-5p, -659-3p, and -3917) were significantly overexpressed in the primary tumors from PM-positive patients in the testing cohort (p=0.002, 0.04 and 0.007 respectively), and distinguished patients with vs. without peritoneal metastasis with the value of area under the curve (AUC) of 0.82. Furthermore, high expression of these miRNAs also associated with poor prognosis (HR=2.18, p=0.04). The efficacy of the combination miRNA-signature was subsequently validated in an independent validation cohort (AUC=0.74). Finally, our miRNA signature when combined together with the macroscopic Borrmann’s type score offered a much superior diagnostic in all three cohorts (AUC=0.87, 0.76, 0.79, respectively), and led us to establish a risk-prediction nomogram for the diagnosis of PM in GC patients. CONCLUSIONS: We have established a miRNA-based signature that have a potential to identify peritoneal metastasis in GC patients.

BACKGROUND & AIMS: Although identification of lymph node (LN) metastasis is a well-recognized strategy for improving outcomes in patients with gastric cancer (GC), currently there is lack of availability of adequate molecular biomarkers that can identify such metastasis. Herein we have developed a robust gene-expression signature for detecting LN metastasis in early stage GC by using a transcriptomic-wide biomarker discovery and subsequent validation in multiple clinical cohorts. METHODS: A total of 532 patients with pathological T1 and T2 GC from 4 different cohorts were analyzed. Two independent datasets (n=96, and n=188) were used to establish a gene signature for the identification of LN metastasis in GC patients. The diagnostic performance of our gene-expression signature was subsequently assessed in two independent clinical cohorts using qRT-PCR assays (n = 101, and n = 147), and subsequently compared against conventional tumor markers and image-based diagnostics. RESULTS: We established a 15-gene signature by analyzing multiple high throughput datasets, which robustly distinguished LN status in both testing (AUC = 0.765, 95% CI 0.667 - 0.863) and validation cohorts (AUC = 0.742, 95% CI 0.630 - 0.852). Notably, the 15-gene signature was significantly superior compared to the conventional tumor markers, CEA (P = 0.04) and CA19-9 (P = 0.005), as well as computed tomography-based imaging (P = 0.04). CONCLUSIONS: We have established and validated a 15-gene signature for detecting LN metastasis in GC patients, which offers a robust diagnostic tool for potentially improving treatment outcomes in gastric cancer patients. FUNDING STATEMENT: The present work was supported by the grants CA72851, CA181572, CA184792, CA202797 and CA187956 from the National Cancer Institute; a grant (RP140784) from the Cancer Prevention Research Institute of Texas (CPRIT), pilot grants from the Baylor Sammons Cancer Center, as well as funds from the Baylor Scott & White Research Institute awarded to Ajay Goel, and a VPRT grant (9610337) from the City University of Hong Kong, grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. CityU 21101115, 11102317, 11103718), a grant from The Science Technology and Innovation Committee of Shenzhen Municipality (JCYJ20170307091256048) awarded to Xin Wang, and was partially supported by the Shenzhen Research Institute, City University of Hong Kong. DECLARATION OF INTERESTS: None of the authors has any potential conflicts to disclose. ETHICS APPROVAL STATEMENT: Written informed consent was obtained from all patients, and the study was approved by the institutional review boards of all the participating institutions.

Accumulating evidence suggests that cancer cells with stem cell-like features have higher resistance to chemotherapeutic agents. Herein, we identified T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1) as one of the Wnt-signaling associated genes which drives self-renewal and its expression is upregulated by cancer associated fibroblasts (CAFs). TIAM1 expression was assessed in resected colorectal cancer (CRC) tissues from 300 patients who did or did not respond to chemotherapy. siRNA and CRISPR/Cas9 was used to examine whether the inhibition of TIAM1 affects chemosensitivity of CRC. We demonstrate that stemness through Wnt signaling regulates chemosensitivity and this phenomenon occurs exclusively in cancer stem cells. Subsequently, we established patient-derived CAFs and tested whether the drug sensitivity of CRC cell lines is altered with CAF-derived conditioned medium. High-TIAM1 expression correlated significantly with poor prognosis of CRC patients, and was overexpressed in patients who did not respond to chemotherapy. We demonstrated that the inhibition of TIAM1 enhanced sensitivity to chemotherapeutic drugs and reduced tumor invasiveness in a series of experiments in vitro. Moreover, CAF-derived conditioned media increased stemness and chemoresistance in CRC cell lines through TIAM1 overexpression. In addition, we validated TIAM1 associated drug sensitivity using a xenograft model. We have demonstrated that TIAM1 is overexpressed in CRC tumors from patients who did not respond to chemotherapeutic drugs and levels of TIAM1 expression served as an independent prognostic factor. Mechanistically, CAFs enhanced CRC chemoresistance through TIAM1 overexpression. Collectively, these results suggest that TIAM1 regulates chemosensitivity in tumors and stroma and thus may be an attractive therapeutic target.

Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD). There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here, we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulating mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and validation cohorts with biopsy-confirmed NAFLD (n = 176 and 59, respectively) and healthy subjects (n = 23), we performed serum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways reflective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fibrosis (F2–F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probability of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clinical practice. NEW & NOTEWORTHY This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to develop an NAFLD diagnostic classifier.

Background and Aims: Hematologic malignancies account for approximately 10% of all cancers in the US. In order to improve the treatment and outcome of patients with hematologic malignancies, integration of accurate noninvasive molecular biomarkers to diagnose, monitor therapeutic response and identify disease subtype are critical. Recent advances in next generation sequencing (NGS) have accelerated the utilization of circulating nucleic acids for the development of non-invasive disease biomarkers. We have developed an RNA-Seq platform that allows robust quantification of the circulating cell-free messenger RNA (cf-mRNA) whole transcriptome. Here, we conducted cf-mRNA profiling on plasma samples collected from multiple myeloma and acute myeloid leukemia patients to investigate the feasibility of cell-free transcriptomic profiling as a monitoring tool for hematologic malignancies following bone marrow transplantation and therapeutic treatment. Methods: We performed cf-mRNA RNA-Sequencing on 96 longitudinally collected plasma samples from 3 multiple myeloma and 3 acute myeloid leukemia patients. Cf-mRNA profiles of patients were evaluated using GTEx (tissue type) and Blueprint (cell type), and dysregulation of gene-expression signatures were assessed following bone marrow transplantation and therapeutic treatment. In addition, we sequenced corresponding whole blood cells and buffy coat for comparison. Results: Plasma cf-mRNA profiling showed substantially higher proportion of transcripts from megakaryocyte and non-hematopoietic cells relative to whole blood cellular transcripts, suggesting that the molecular profile of cf-mRNA is more suitable for the assessment of gene-expression dysregulation of bone marrow than whole blood. We further report that non-invasive cf-mRNA profiling captures gene-expression alterations of cancer patients following chemotherapy and bone marrow transplantation. And we observed dysregulation of hematopoietic lineage-specific transcripts in circulation during bone marrow ablation and reconstitution. Lastly, we used cf-mRNA profiling to identify a malignant Ig clone for multiple myeloma patients and were able to monitor bone marrow reconstitution. Conclusions: We demonstrated that cf-mRNA profiling is an effective means to non-invasively assess molecular characteristics that are associated with multiple clinical parameters of hematological malignancies. Our study highlights the potential of cf-mRNA Seq as a platform to develop patient monitoring biomarkers for hematologic malignancies. Citation Format: Shusuke Toden, David A. Ross, Stephen R. Quake, John J. Sninsky. Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5174.

Transcriptomic profiles of Alzheimer’s disease patients and healthy individuals were investigated using cell-free mRNA sequencing., The lack of accessible noninvasive tools to examine the molecular alterations occurring in the brain limits our understanding of the causes and progression of Alzheimer’s disease (AD), as well as the identification of effective therapeutic strategies. Here, we conducted a comprehensive profiling of circulating, cell-free messenger RNA (cf-mRNA) in plasma of 126 patients with AD and 116 healthy controls of similar age. We identified 2591 dysregulated genes in the cf-mRNA of patients with AD, which are enriched in biological processes well known to be associated with AD. Dysregulated genes included brain-specific genes and resembled those identified to be dysregulated in postmortem AD brain tissue. Furthermore, we identified disease-relevant circulating gene transcripts that correlated with the severity of cognitive impairment. These data highlight the potential of high-throughput cf-mRNA sequencing to evaluate AD-related pathophysiological alterations in the brain, leading to precision healthcare solutions that could improve AD patient management.

Recent advances have begun to clarify the physiological and pathological roles of non-coding RNAs (ncRNAs) in various diseases, including cancer. Among these, microRNAs (miRNAs) have been the most studied and have emerged as key players that are involved in the regulation of important growth regulatory pathways in cancer pathogenesis. The ability of a single ncRNA to modulate the expression of multiple downstream gene targets and associated pathways, have provided a rationale to pursue them for therapeutic drug development in cancer. In this context, early data from pre-clinical studies have demonstrated that synthetic miRNA-based therapeutic molecules, along with various protective coating approaches, has allowed for their efficient delivery and anti-tumor activity. In fact, some of the miRNA-based cancer therapeutic strategies have shown promising results even in early-phase human clinical trials. While the enthusiasm for ncRNA-based cancer therapeutics continue to evolve, the field is still in the midst of unraveling a more precise understanding of the molecular mechanisms and specific downstream therapeutic targets of other lesser studied ncRNAs such as the long-non-coding RNAs, transfer RNAs, circular RNAs, small nucleolar RNAs, and piwi-interacting RNAs. This review article provides the current state of knowledge and the evolving principles for ncRNA-based therapeutic approaches in cancer, and specifically highlights the importance of data to date and the approaches that are being developed to overcome the challenges associated with their delivery and mitigating the off-target effects in human cancers.

Additional file 2: Supplementary Table 2–7

Additional file 3: Methods

Proanthocyanidins are a heterogeneous group of flavan-3-ol or flavan-3,4-diol oligomers present in various fruits and vegetables. In particular, the smaller oligomeric subset of proanthocyanidins, termed the oligomeric proanthocyanidins (OPCs) appear to have potent anti-tumorigenic properties, but the underlying mechanisms for their effectiveness remain unclear. Herein, we utilized a series of in vitro, in vivo and patient-derived organoid approaches to systematically investigate the chemoprotective role of OPCs in colorectal cancer. OPCs exerted anti-tumorigenic effects through inhibition of cellular proliferation, and induced apoptosis and cell cycle arrest. Intriguingly, OPCs suppressed spheroid derived cancer stem-like cell formation and decreased the expression of intestinal cancer stem cell markers including LGR5, CD44 and CD133. Mechanistically, RNA-sequencing results confirmed that OPCs prominently interfered with developmental and self-renewal pathways and identified several self-renewal associated oncogenes targeted by OPCs. Furthermore, OPCs inhibited Hippo pathway through downregulation of its key transcriptional regulators, YAP and TAZ. Finally, we confirmed anti-tumorigenic effects of OPCs using multiple xenograft experiments and recapitulated its protective effects using patient-derived colorectal tumor organoids. Collectively, we have comprehensively assessed anti-tumorigenic properties of OPCs and our data throws light on previously unrecognized chemopreventive mechanisms of OPCs highlighting its therapeutic potential.

Background Emerging evidence indicates that small nucleolar RNAs (snoRNAs) play a central role in oncogenesis. Herein, we systematically evaluated expression profiles of snoRNAs in colorectal cancer (CRC) and investigated their clinical and functional role in this malignancy. Methods We compared expression levels of snoRNAs between cancer and normal tissues using publicly available datasets and identified the most differentially expressed and commonly upregulated snoRNAs in CRC. These results were examined in 489 colorectal tissues to assess their clinical significance, followed by a series of in vitro and in vivo experiments to evaluate the functional role of candidate snoRNAs. Results Using multiple RNA profiling datasets, we identified consistent overexpression of SNORA21 in CRC. In the clinical validation cohorts, the expression level of SNORA21 was upregulated in colorectal adenomas and cancers. Furthermore, elevated SNORA21 emerged as an independent factor for predicting poor survival. Both in vitro and in vivo experiments revealed that CRISPR/Cas9-mediated inhibition of SNORA21 expression resulted in decreased cell proliferation and invasion through modulation of multiple cancer related pathways. Conclusions We systematically identified SNORA21 as a key oncogenic snoRNA in CRC, which plays an important role in cancer progression, and might serve as an important prognostic biomarker in CRC., Highlights • Systematic and comprehensive analysis revealed that SNORA21 was significantly upregulated in CRC. • Elevated SNONA21 expression was related to metastasis and predicted poor prognosis in CRC patients. • SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC. Emerging evidence indicates that small nucleolar RNAs (snoRNAs) might play a central role in oncogenesis; however, the clinical significance and functional roles of snoRNAs in colorectal cancer (CRC) remain unclear. Herein, using multiple publicly available datasets, we systematically assessed expression profiles of snoRNAs in CRC and elucidated that the high expression of SNORA21 was associated with distant metastasis and poor overall survival. A series of in vitro and in vivo experiments revealed oncogenic role for SNORA21. Our findings indicate that SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients. Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings. Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.