The long noncoding RNA lnc-FANCI-2 intrinsically restricts RAS signaling and phosphorylation of Akt and Erk in HPV16-infected cervical cancer
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eLife Assessment
This important study reports new insights into the roles of a long noncoding RNA, lnc-FANCI-2, in the progression of cervical cancer induced by a type of human papillomavirus. Through a blend of cell biological, biochemical, and genetic analyses of RNA and protein expression, protein-protein interaction, cell signaling, and cell morphology, the authors provide convincing evidence that lnc-FANCI-2 affects cervical cancer outcome by regulating the RAG signaling pathway. These findings will be of interest to scientists in the fields of cervical cancer, long noncoding RNA, and cell signaling.
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Abstract
We recently discovered increased expression of a long noncoding RNA, lnc-FANCI-2, coinciding with cervical lesion progression from CIN1, CIN2-3 to cervical cancer. Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. To explore possible roles of lnc-FANCI-2 in HPV-induced cervical carcinogenesis, we ablated the expression of lnc-FANCI-2 in the HPV16-positive cervical cancer cell line, CaSki. Knock-out (KO) single cell clones expressed HPV16 oncogenes normally but displayed altered cell morphology when compared with the parental cells. Proteomic profiling of cytosolic and secreted proteins from the parental and KO cells showed that lnc-FANCI-2 regulates expression of a subset of cell surface and adhesion-related proteins, including inhibition of MCAM, PODXL2 and ECM1 and increased levels of ADAM8 and TIMP2. RNA-seq analyses revealed that, relative to the parental cells, KO cells exhibited significantly increased RAS signaling but decreased IFN pathways. In KO cells, phosphorylated Akt and Erk1/2, two important RAS pathway effectors, were increased more than 3-fold, accompanied by increase of IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decrease of RAC3. Accordingly, high levels of lnc-FANCI-2 and lower levels of MCAM in cervical cancer patients are associated with improved survival. We found that lnc-FANCI-2 in CaSki cells interacts specifically with 32 host proteins, including H13, HNRH1, K1H1, MAP4K4, and RNPS1, and knockdown of MAP4K4 led to increase phosphorylation of Akt and Erk1/2. In summary, a key function of lnc-FANCI-2 is to intrinsically regulate RAS signaling, thereby affecting cervical cancer outcome.
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eLife Assessment
This important study reports new insights into the roles of a long noncoding RNA, lnc-FANCI-2, in the progression of cervical cancer induced by a type of human papillomavirus. Through a blend of cell biological, biochemical, and genetic analyses of RNA and protein expression, protein-protein interaction, cell signaling, and cell morphology, the authors provide convincing evidence that lnc-FANCI-2 affects cervical cancer outcome by regulating the RAG signaling pathway. These findings will be of interest to scientists in the fields of cervical cancer, long noncoding RNA, and cell signaling.
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Reviewer #1 (Public review):
Summary:
The authors attempted to dissect the function of a long non-coding RNA, lnc-FANCI-2, in cervical cancer. They profiled lnc-FANCI-2 in different cell lines and tissues, generated knockout cell lines, and characterized the gene using multiple assays.
Strengths:
A large body of experimental data has been presented and can serve as a useful resource for the scientific community, including transcriptomics and proteomics datasets. The reported results also span different parts of the regulatory network and open up multiple avenues for future research.
Weaknesses:
The write-up is somewhat unfocused and lacks deep mechanistic insights in some places.
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Reviewer #2 (Public review):
The study by Liu et al provides a functional analysis of lnc-FANCI-2 in cervical carcinogenesis, building on their previous discovery of FANCI-2 being upregulated in cervical cancer by HPV E7.
The authors conducted a comprehensive investigation by knocking out (KO) FANCI-2 in CaSki cells and assessing viral gene expression, cellular morphology, altered protein expression and secretion, altered RNA expression through RNA sequencing (verification of which by RT-PCR is well appreciated), protein binding, etc. Verification experiments by RT-PCR, Western blot, etc are notable strengths of the study.
The KO and KD were related to increased Ras signaling and EMT and reduced IFN-y/a responses.
Although the large amount of data is well acknowledged, it is a limitation that most data come from CaSki cells, in which …
Reviewer #2 (Public review):
The study by Liu et al provides a functional analysis of lnc-FANCI-2 in cervical carcinogenesis, building on their previous discovery of FANCI-2 being upregulated in cervical cancer by HPV E7.
The authors conducted a comprehensive investigation by knocking out (KO) FANCI-2 in CaSki cells and assessing viral gene expression, cellular morphology, altered protein expression and secretion, altered RNA expression through RNA sequencing (verification of which by RT-PCR is well appreciated), protein binding, etc. Verification experiments by RT-PCR, Western blot, etc are notable strengths of the study.
The KO and KD were related to increased Ras signaling and EMT and reduced IFN-y/a responses.
Although the large amount of data is well acknowledged, it is a limitation that most data come from CaSki cells, in which FANCI-2 localization is different from SiHa cells and cancer tissues (Figure 1). The cytoplasmic versus nuclear localization is somewhat puzzling.
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Reviewer #3 (Public review):
Summary:
A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.
Strengths:
(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and …
Reviewer #3 (Public review):
Summary:
A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.
Strengths:
(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further understanding of other cellular alterations in HPV oncogenesis.
(2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines, and HPV transduced cell lines.
(3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.
(4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2, and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.
(5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.
(6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.
Weaknesses:
(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2.
(2) Previous studies and data in the current showed a steadily increased Inc-FANCI-2 during cancer progression, however, the authors did not observe significant changes in cell behaviors (both morphology and proliferation) in KO Inc-FANCI-2.
(3) The authors observed the significant changes of RAS signaling (downstream) in KO cells, but they provided limited interpretations of how these results contributed to full transformation or tumorigenesis in HPV-positive cancer.
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