Long Noncoding RNAs in Human Papillomavirus-associated Pathogenesis

Long Noncoding RNAs in Human Papillomavirus-associated Pathogenesis: Comparison

Please note this is a comparison between Version 1 by Karl Munger and Version 2 by Peter Tang.

Papillomaviruses are a large family of non-enveloped viruses with ~8000 base pair, circular, double stranded DNA genomes. They have been detected in almost all vertebrates, are highly host-specific and preferentially infect squamous epithelial tissues. Infections with high-risk human papillomaviruses cause ~5% of all human cancers. E6 and E7 are the only viral genes that are consistently expressed in cancers, and they are necessary for tumor initiation, progression, and maintenance. E6 and E7 encode small proteins that lack intrinsic enzymatic activities and they function by binding to cellular regulatory molecules, thereby subverting normal cellular homeostasis. Much effort has focused on identifying protein targets of the E6 and E7 proteins, but it has been estimated that ~98% of the human transcriptome does not encode proteins. Long noncoding RNAs (lncRNAs) are defined as transcripts of >200 nucleotides with no or limited coding potential of <100 amino acids. There is a growing interest in studying noncoding RNAs as biochemical targets and biological mediators of human papillomavirus (HPV) E6/E7 oncogenic activities.

human papillomavirus
viral oncogenesis
cervical carcinoma
lncRNA
E6
E7

1. Human Papillomaviruses as Oncogenic Drivers

Papillomaviruses are a large family of non-enveloped viruses with ~8000 base pair, circular, double-stranded DNA genomes. They have been detected in almost all vertebrates, are highly host-specific, and preferentially infect squamous epithelial tissues. More than 440 human papillomaviruses (HPVs) have been molecularly characterized as of 03/2020, and they are organized into five phylogenetic genera: alpha, beta, gamma, mu, and nu [1]. HPVs exhibit a marked preference for infecting specific squamous epithelial tissue types; most alpha HPVs infect mucosal epithelia, whereas beta, gamma, mu, and nu HPVs preferentially infect cutaneous epithelia. HPV infections are either asymptomatic or cause the formation of generally benign hyperplastic lesions, commonly referred to as warts. Some cutaneous HPV infections contribute to the initiation of cutaneous squamous cell carcinomas, particularly in long-term immunosuppressed organ transplant patients, and in individuals with a rare hereditary skin disease, epidermodysplasia verruciformis ^[2][3][2,3]. The mucosal alpha HPVs can be clinically classified into low and high-risk types. Low-risk HPVs cause benign genital warts, whereas high-risk HPVs cause premalignant lesions that can progress to carcinomas. Approximately 5% of all human cancers are caused by high-risk HPV infections. These include almost all cervical carcinomas, a large fraction of other anogenital tract carcinomas, and a growing percentage of oral cancers, particularly oropharyngeal carcinomas [4].

High-risk HPV-associated cancers are generally non-productive infections and only two viral genes, E6 and E7, are consistently expressed. HPV E6 and E7 encode low molecular weight, cysteine-rich, zinc-binding proteins of ~150 and ~100 amino acids, respectively. Despite their diminutive size, they are potent oncogenic drivers and are necessary for tumor initiation, progression, and maintenance. They lack intrinsic enzymatic activities and do not directly bind to specific DNA sequences. Hence, they function by binding to host cellular regulatory molecules, thereby subverting their normal physiological activities ^[5][6][5,6]. As a consequence, HPV E6 and E7 target almost all cellular processes that have been designated “hallmarks of cancer” ^[7][8][7,8]. A large number of cellular protein interaction targets for E6 and E7 have been identified, most prominently the TP53 and retinoblastoma (RB1) tumor suppressor proteins, respectively ^[9][10][9,10].

2. Long Noncoding RNAs

Long noncoding RNAs (lncRNAs) are defined as transcripts of >200 nucleotides with no or limited coding potential of <100 amino acids. Large intergenic noncoding RNAs (lincRNAs) are a subset of lncRNAs that do not overlap with protein-coding genes, whereas other lncRNAs share some overlap, either on the sense or antisense strand, with coding genes ^[11][12]. The first cellular lncRNAs, H19 and X-Inactive Specific Transcript (XIST), were discovered in the early 1990s ^[12][13][13,14]. With the development of high-throughput sequencing techniques in the late 2000s, there was a substantial increase in identified lncRNAs. Compared to the ~21,000 protein-coding genes, the number of lncRNA genes has been estimated to be in the range of ~15,000 to ~58,000 ^[14][15][15,16]. As sequencing depth increases, it is expected that additional lncRNAs will be identified. The majority of lncRNAs are transcribed by RNA Polymerase II, have 5′ cap structures, and are 3′ polyadenylated, rendering them biochemically indistinguishable from mRNAs. LncRNAs can localize to nuclear as well as cytoplasmic compartments.

Only ~20% of lncRNA nucleic acid sequences are significantly conserved between humans and mice, whereas the remaining lncRNAs only share small areas of microhomology ^[16][17]. The fact that such microhomologies are significant has been impressively demonstrated by complementation experiments. For example, despite limited sequence similarity of the linc-birc6 (megamind) and linc-oip5 (cyrano) lncRNAs across species, the phenotype of megamind and cyrano depletion in zebrafish was rescued by expression of murine or human transgenes ^[17][18].

LncRNAs can interact with linear RNA or DNA sequences by base pairing. Moreover, secondary and tertiary lncRNA structures can also act as recognition surfaces for binding proteins with high affinity and specificity. Molecular interactions with RNA, DNA and proteins furnish almost endless possibilities for lncRNAs modes of action. These include epigenetic regulation of gene expression, forming scaffolds for macromolecular complex assembly, binding, and inactivation of miRNAs (“sponging”), and regulating mRNA stability (Figure 1).

Figure 1.

Major mechanisms of action of long noncoding RNAs (lncRNAs). See text for detail.

The role of nuclear lncRNAs in epigenetic regulation has been extensively investigated, and there are numerous examples of lncRNAs affecting the epigenetic status of neighboring loci (in cis) or at distant loci (in trans). A classic example of a lncRNA acting in cis is the X-inactive specific transcript (XIST). During X-inactivation, XIST accumulates in cis where it tethers polycomb repressive complexes to silence genes on the X-chromosome, a phenomenon referred to as X-inactivation ^[18][19]. The HOX transcript antisense intergenic RNA (HOTAIR), transcribed from the HOXC locus, acts in trans by guiding chromatin repressive complexes to HOXD and other chromosomal loci ^[19][20]. Other lncRNAs such as the HOXA transcript at the distal tip (HOTTIP) and nettoie Salmonella pas Theiler’s (NeST) cause activation of target genes by recruiting WDR5, a component of the MLL/MLL1 histone H3 lysine 4 methyltransferase complex, which marks genes for transcriptional activation ^[20][21][21,22].

The ability of nuclear or cytoplasmic lncRNA to associate with proteins allows them to function as scaffolds for the assembly of individual proteins into functional complexes. The nuclear enriched abundant transcript 1 (NEAT1) lncRNA, for example, forms a complex with the HEXIM1 protein to assemble a complex that contains DNAPK, cGAS, TBK1 and IRF3, which is necessary to trigger innate immune signaling in response to cytoplasmic DNA sensing ^[22][23].

Cytoplasmic lncRNAs have also been reported to act as “miRNA sponges”. By base pairing with individual microRNAs they can restrain their abilities to bind to and inhibit their mRNA targets, thereby interfering with miRNA mediated repression ^[23][24].

Lastly, cytoplasmic lncRNAs can directly or indirectly bind mRNAs thereby modulating their stability and/or translation. The pro-differentiation terminal differentiation-induced lncRNA (TINCR), for example, binds and stabilizes mRNAs that are critical for epithelial differentiation through the recruitment of the Staufen RNA binding protein ^[24][25].

Given the versatility of their biochemical modes of action, it comes as no surprise that cellular lncRNA expression is dysregulated in many cancers. However, there have been only very few studies that have carefully evaluated how specific, well-established oncogenic drivers trigger dysregulation of lncRNA expression and how this may contribute to carcinogenesis. Given that HPV E6 and E7 are universal drivers of ~5% of human cancers, they are ideally suited to address this critical matter.

3. Deregulation of lncRNAs in Cervical Carcinomas

There have been numerous studies reporting increased (Table 1) or decreased (Table 2) expression of specific lncRNAs in HPV-associated premalignant lesions and cancers (see tables below for references). By proposing specific mechanisms of action and linking aberrant expression to specific oncogenic phenotypes, these studies suggest that dysregulated lncRNA expression may importantly contribute to HPV carcinogenesis by subverting cellular processes that have been referred to as “hallmarks of cancer” ^[7][25][7,26].

Table 1.

lncRNAs reported to be upregulated in various models of cervical lesions and cancers.

lncRNA

Table 2.

lncRNAs reported to be downregulated in various models of cervical lesions and cancers.

lncRNA	Oncogenic Phenotype	Oncogenic Phenotype Proposed Mechanism	References
	Proposed Mechanism		Reference
ANRIL
GAS5	Proliferation, migration, invasion		Proliferation, invasion, migration PI3K/AKT; Cyclin D1, CDK4, CDK6, N-cadherin, Vimentin expression	E-cadherin, Vimentin ^[26]^[27][27,28]
	^[	⁷⁵	^][76]	ARAP1-AS1	Proliferation, invasion	MYC translation by PSF/PTB
	Proliferation, migration, invasion, colony formation	miR-21 expression/STAT3	^[28][29]
	^[	⁷⁶	^][77]	BLACAT1	Proliferation, migration, invasion
	Radiosensitivity	WNT signaling/β-catenin	^[29][30]
	miR-106b sponging/IER3		^[77][78]	CCAT2	Proliferation, apoptosis	None reported	^[
HOTAIR	Decreased polycomb repression	Binding to HPV E7	^30][31]
	^[	⁷⁸	^][79]	CCEPR (CCHE1)
Lnc-CCDST	Proliferation	PCNA mRNA stabilization	^[31	Migration, invasion, angiogenesis ^][32]
	DHX9, MDM2 scaffolding		^[79][80]
MEG3	Proliferation	Proliferation, apoptosis independent of PCNA mRNA		Binding, degradation of P-STAT3 ^[32][33]
	^[	⁸⁰	^][81]	CRNDE	Proliferation, migration, invasion
	Proliferation, colony formation, apoptosis	miR-183 sponging/cyclin B1		miR-21-5p expression/TP53 ^[33][34]
	^[	⁸¹	^][82]		Proliferation
STXBP5-AS1		PUMA expression	Viability, invasion ^[34][35]
	miR-96-5p expression/PTEN		^[82][83]	DANCR	Proliferation, migration, invasion
TINCR		miR-665 sponging/TGFβ-R1-ERK-SMAD	Differentiation, colony formation, migration ^[35][	S100A8 and other ZNF750 targets 36]
	^[	⁸³	^][84]
WT1-AS	Proliferation, migration, invasion, epithelial to mesenchymal transition (EMT)		Proliferation miR-335-5p sponging/ROCK1	TP53 ^[36][37]
		^[	^84][85]	EBIC (TMPOP2)	Motility, invasion	E-cadherin silencing by EZH2	^[37
	Proliferation, invasion, migration	^]	[	miR-203a-5p binding/FOXN2 38]
	^[	⁸⁵	^][86]		Proliferation	miR-375, miR-139 sponging HPV E6/E7 expression	^[38][39]
XLOC_010588	Proliferation	MYC mRNA binding/degradation	^[86][87]	FAM83H-AS1	Proliferation, migration and apoptosis	G1/S-phase transition	^[39][40]
GATA6-AS	Migration, invasion	MTK-1	^[40][41]
H19	Proliferation, anchorage independent growth	None reported	^[41][42]
HOTAIR	Apoptosis, invasion, migration	NOTCH signaling	^[42][43]
	Apoptosis, proliferation, invasion	miR-23b sponging/MAPK1 axis	^[43][44]
	Autophagy, EMT	WNT signaling	^[44][45]
	Proliferation	miR-143-3p sponging/BCL2	^[45][46]
HOXD-AS1	Proliferation	Ras/ERK	^[46][47]
Linc00483	Proliferation, apoptosis, invasion, migration	miR-508-3p sponging/RGS17	^[47][48]
LINP1	DNA damage repair (Non-homologous end joining)	KU80, DNA-PKcs binding	^[48][49]
Lnc-IL7R	Apoptosis	BCL2/caspase 3	^[49][50]
LUCAT1	Proliferation, migration, invasion	miR-181a sponging	^[50][51]
MALAT1	Cell invasion and metastasis	inhibition of EMT genes	^[51][52]
	Proliferation, migration, invasion	miR-625-5p/AKT2	^[52][53]
	Proliferation	Mir-625-5p/NF-kB signaling	^[53][54]
	Cisplatin resistance	PI3K/AKT	^[54][55]
MIR205HG	Proliferation, apoptosis, migration	SRSF1/KRT17 axis	^[55][56]
NEAT1	Proliferation, invasion	PI3K/AKT	^[56]^[57][57,58]
	Colony formation, migration, invasion	miR-133a sponging/SOX4	^[58][59]
NORAD	Proliferation, invasion	miR-590-3p sponging/SIP1	^[59][60]
PANDAR	Proliferation	None reported	^[60][61]
PVT1	Proliferation, invasion	Inhibiting TGFβ; miR-140-5p sponging/SMAD3	^[61]^[62][62,63]
	EMT, chemoresistance	miR-195 epigenetic silencing	^[63][64]
SNHG8	Proliferation, apoptosis	RECK silencing by EZH2	^[64][65]
SNHG12	Proliferation, apoptosis	ERK/Slug	^[65][66]
SNHG16	Proliferation, invasion	PARP9 expression by SPI1 binding	^[66][67]
TUG1	Proliferation, apoptosis, invasion, tumor growth	miR-138-5p sponging/SIRT1	^[67][68]
	Proliferation, apoptosis, EMT	BCL-2, caspase 3; fibronectin, vimentin, and cytokeratin	^[68][69]
TP73-AS1	Proliferation, migration	miR-329-3p sponging/SMAD2	^[69][70]
	Proliferation, migration, invasion	miR-607 sponging/CCND2	^[70][71]
UCA1	Radioresistance	HK2/glycolytic pathway	^[71][72]
XIST	Proliferation	miR-140-5p sponging/ORC1	^[72][73]
	Proliferation, invasion, apoptosis, EMT	miR-200a sponging/FUS	^[73][74]
ZEB-AS1	Proliferation, migration, invasion, EMT	ZEB1 expression	^[74][75]

4. Deregulation of lncRNAs by HPV E6 and/or E7 Proteins

Several reviews have focused on the clinical implications of lncRNA expression changes in HPV-associated cancers ^[87][88][89][88,89,90], but dysregulation of cellular lncRNA expression in HPV-associated lesions and cancers does not infer that the observed changes represent a primary consequence of HPV infection and E6 and/or E7 expression. Some of the studies cited in the tables above implicated E6 and/or E7 as regulators of certain lncRNAs, including PVT1, MALAT1, SNHG12, lnc-CCDST, LINC01101, and LINC00277 ^{[63][65][79][90][91]}[64,66,80,91,92] by depleting E6/E7 expression in cervical cancer lines.

To determine how HPV16 E6/E7 expression deregulates lncRNA expression in normal human epithelial cells, thwe researchers aanalyzed RNA sequencing (RNAseq) data of two independently derived populations of HPV16 E6/E7 expressing primary human foreskin keratinocytes (HFKs) and their donor and passage matched, control vector-transduced parental cells ^[92][93]. Of the 7109 annotated lncRNA species that were detectably expressed, the levels of 1453 were altered at least twofold. Of these, 1070 lncRNAs were expressed at higher levels whereas 383 were expressed at lower levels in HPV16 E6/E7 expressing HFKs than in parental HFKs (Figure 2A).

Figure 2. Differential expression of cellular lncRNAs in human papillomavirus (HPV)16 E6/E7 expressing human foreskin keratinocytes (HFKs). (A). Expression of annotated cellular lncRNA by RNA sequencing (RNAseq) analysis of HPV16 E6/E7 expressing and parental primary HFKs. (B). Quantitative reverse transcription PCR (qRT-PCR) analysis of select cellular lncRNAs in HPV16 E6/E7 expressing versus parental HFKs. See text for detail.

From this list, the rwesearchers analyzed by quantitative reverse transcription PCR (qRT-PCR), expression of a small number of lncRNAs that were shown to be dysregulated in HPV-associated lesions and cancers (see Table 1; Table 2) or are well-established modulators of cancer hallmarks targeted by HPV16 E6/E7. From this panel, the most significantly upregulated and downregulated lncRNAs are the cervical carcinoma expressed PCNA regulatory lncRNA (CCEPR) and the DNA damage-induced noncoding lncRNA (DINO), respectively. HOTAIR, human ovarian cancer-specific transcript 2 (HOST2), growth arrest-specific 5 (GAS5), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and tissue differentiation-inducing non-protein coding RNA (TINCR) were downregulated, whereas hepatocellular carcinoma up-regulated EZH2-associated lncRNA (HEIH), differentiation antagonizing non-protein coding RNA (DANCR), EZH2-binding lncRNA in cervical cancer (EBIC), neuroblastoma associated transcript 1 (NBAT1) and H19 were upregulated in HPV16 E6/E7 expressing HFKs (Figure 2B).