Cancer spread to lymph nodes predicts poor survival but underlying mechanisms remain little understood. In this study, we show that overexpression of the long noncoding RNA LNMICC associates with lymph node metastasis of primary cervical cancer, where it serves as an independent high-risk factor in patient survival. Functional investigations demonstrated that LNMICC promoted lymph node metastasis by reprogramming fatty acid metabolism, by recruiting the nuclear factor NPM1 to the promoter of the fatty acid binding protein FABP5. We also found that the prometastatic effects of LNMICC were directly targeted and suppressed by miR-190. Our results establish a new mechanism of lymph node metastasis and highlight LNMICC as a candidate prognostic biomarker and therapeutic target in cervical cancer. Significance: These results establish the role of a novel long noncoding RNA in lymph node metastasis, with implications as a candidate prognostic biomarker and therapeutic target in cervical cancer. Cancer Res; 78(4); 877–90. ©2017 AACR.

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro‐metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin⁺CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin⁺CAFs impaired lymphatic endothelial barriers by activating the integrin‐FAK/Src‐VE‐cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin‐induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin^‐CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF‐enriched cases without LNM. In conclusion, we identified a specific periostin⁺CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast‐targeted interventions that block CAF‐dependent metastasis.

Integration of human papillomavirus (HPV) DNA into the human genome is a reputed key driver of cervical cancer. However, the effects of HPV integration on chromatin structural organization and gene expression are largely unknown. We studied a cohort of 61 samples and identified an integration hot spot in the CCDC106 gene on chromosome 19. We then selected fresh cancer tissue that contained the unique integration loci at CCDC106 with no HPV episomal DNA and performed whole-genome, RNA, chromatin immunoprecipitation and high-throughput chromosome conformation capture (Hi-C) sequencing to identify the mechanisms of HPV integration in cervical carcinogenesis. Molecular analyses indicated that chromosome 19 exhibited significant genomic variation and differential expression densities, with correlation found between three-dimensional (3D) structural change and gene expression. Importantly, HPV integration divided one topologically associated domain (TAD) into two smaller TADs and hijacked an enhancer from PEG3 to CCDC106, with a decrease in PEG3 expression and an increase in CCDC106 expression. This expression dysregulation was further confirmed using 10 samples from our cohort, which exhibited the same HPV-CCDC106 integration. In summary, we found that HPV-CCDC106 integration altered local chromosome architecture and hijacked an enhancer via 3D genome structure remodeling. Thus, this study provides insight into the 3D structural mechanism underlying HPV integration in cervical carcinogenesis.

Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287–1,856), and the specificity could be reversely correlated with the counts of middle “G” in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/αN/βN) and G recognition modules (NN/NH) and found the design (αN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo off-target detection pipeline for three generations of artificial nucleases provided useful tools for genome engineering-based gene therapy.

Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is still poorly understood. In this study, we employed integrative analysis on multi-omics data of six HPV-positive and three HPV-negative cell lines. Through HPV integration detection, super-enhancer (SE) identification, SE-associated gene expression and extrachromosomal DNA (ecDNA) investigation, we aimed to explore the genome-wide transcriptional influence of HPV integration. We identified seven high-ranking cellular SEs generated by HPV integration in total (the HPV breakpoint-induced cellular SEs, BP-cSEs), leading to intra-chromosomal and inter-chromosomal regulation of chromosomal genes. The pathway analysis revealed that the dysregulated chromosomal genes were correlated to cancer-related pathways. Importantly, we demonstrated that BP-cSEs existed in the HPV–human hybrid ecDNAs, explaining the above transcriptional alterations. Our results suggest that HPV integration generates cellular SEs that function as ecDNA to regulate unconstrained transcription, expanding the tumorigenic mechanism of HPV integration and providing insights for developing new diagnostic and therapeutic strategies.