Cervical Cancer mechanism

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最新推荐文章于 2022-07-31 12:43:30 发布

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HPV感染致癌机理

中国高危HPV型是16，18，52，58，33
在HPV整合的过程中，不仅会破坏病毒本身的基因组，宿主基因结构也会发生改变。
HPV整合时破坏了E2基因的开放阅读框（ORF），导致E2基因表达受抑，从而失去对HPV两个重要的癌基因E6和E7的表达调控，抑制p53和pRB的功能，使得宿主细胞凋亡失控，诱导细胞无限增生，转向恶性。而在某些HPV阳性的恶性肿瘤中，如头颈部鳞癌样本中E6和E7基因的表达水平并非都会增加，这就意味着癌变的发生也有可能来自宿主基因的改变。

HPV整合后宿主`抑癌基因`失活

病毒选择性地插入宿主基因中，同时伴随周期性的区域扩增、切除和重排，这些都会直接影响宿主基因
Loss of function of a tumor surpressor[抑癌基因被HPV整合]
Enhanced expression of an oncogene
1. Application of a nearby region(HPV integrated region，HPV区域附近amp时，原癌基因也amp)
2. Enhanced oncogene expression from viral promoters[hpv整合部分在上游，当作启动子]
3. Interchromosomal rearrangements leading to altered expression of genes in involved regions（染色体内部重组）
Parfenov等人发现原发性HNSCC肿瘤样本中抑癌基因RAD51（DNA修复相关基因）表达产物失去功能。进一步研究发现，HPV病毒整合位于宿主RAD51基因的8号内含子中，整合后8号内含子中约42kb的区域与病毒插入序列同时扩增28倍，扩增后破坏了RAD51原有的转录本，导致表达的蛋白失去活性（如图2）。而Ojesina等人发现多个宫颈癌样本中HPV16、HPV18和HPV52的整合位点也在RAD51基因中，病毒整合后RAD51基因表达产物失活，宿主基因组稳定性大大降低，促进癌症发生发展。
类似的功能失活的抑癌基因还有ETS2基因，虽然病毒整合后基因结构保持完整，拷贝数变化也不大，但整合位点附近的7号和8号外显子表达水平大幅下调，导致基因表达产物功能失活。这也是9号染色体上抑癌基因CD274功能失活的主要原因。

HPV整合可刺激宿主`癌基因`高表达

1，HPV整合插入宿主癌基因的上游，区域扩增时会纳入邻近区域的癌基因。2，宿主癌基因利用病毒的启动子增强表达。
癌基因NR4A2高表达就是其中的典型代表：该基因上游30Kb区域是HPV病毒的整合区，病毒基因及其邻近区域共75Kb为整合时的扩增区,而完整的NR4A2基因就在扩增区域中，病毒在整合中扩增284倍时，NR4A2基因的表达水平也提高了284倍。有趣的是该肿瘤样本中病毒的E6和E7表达水平比较低，也就意味着宿主癌基因高表达同样是肿瘤发生的重要原因（如图3）。Ojesina等人在宫颈癌样本中也有类似发现：FOXE1、PIM1（舌癌细胞）和MY3（宫颈癌样本）等癌基因同样存在高表达现象。

`宿主染色体重排`诱导癌症发生

Parfenov等人在HNSCC样本中发现HPV整合时，宿主的3号和13号染色体中整合位点邻近区域会发生重排（如图4）。重排过程中涉及的基因有3号染色体的TPRG1（肿瘤蛋白p63调节基因1）和TP63以及chr13的KLF5基因。研究发现在染色体重排过程中区域化扩增后，上述重排涉及的基因表达水平显著上升。这些基因中KLF5是调节细胞增殖的转录因子，可促进细胞的增殖；而TP63是鳞状细胞癌中重要的致癌基因，也是上皮细胞生长的转录因子。尽管目前对TPRG1的功能研究尚不透彻，但该基因的高表达同样可能与癌症的发生发展相关。

宿主甲基化模式改变同样是HPV整合所致

如肿瘤抑制基因BARX2和IRX4，及癌基因SIM2 和 CTSE的甲基化模式都发生了改变

Break points related gene(2015 ng article)

AGTR2,DMD,CDH7,DCC,HS3ST4,KLF5,KLF12,HMGA2,CPNE8,DLG2,C9orf85,
POU5F1B,SEMA3D,MSX2,LEPREL1,CADM2,FHIT,LRP1B,FAM84B,MYC,MSRB3

Analysis Identifies Mutations Linked to Cervical Cancer(Cancer Discov. 2014 Mar)

115个宫颈癌tumor-normal样本鉴定出13个复发的基因突变，包括8个未被报道与疾病相关的一级两个未被报道与癌症相关的。转录组数据分析表明HPV病毒整合提升了临近基因的表达。发现了之前鉴定到的发生在宫颈鳞癌的突变，PIK3CA，PTEN，STK11,ERBB2，HER2。

治疗建议
ERBB2，HER2：
The presence of these mutations in some cervical cancers may suggest that these patients would benefit from anti-ERBB2 therapy。
9%的宫颈鳞癌患者发现了HLA-B相关突变，之前的研究报道HLA-A相关突变发生在肺癌中：
These kinds of lung cancer have responded well to some immunotherapies, so the same therapies may eventually be used for some types of cervical cancer as well

新鉴定出的mutated gene包括：EP300(16%), FBXW7(15%), MAPK1(8%), and NFE2L2(4%) in squamous cell carcinoma, and ELF3(13%) and CBFB(8%) in adenocarcinoma.

全外（/WGS 组学）的意义
“For cervical cancer, existing treatments are not very useful,” says Murty Vundavalli, PhD, a cancer geneticist at Columbia University in New York, NY, who did not work on the study. “The main issue should be to identify biomarkers that identify response to specific treatments. Whole-genome studies will ultimately lead to those kinds of approaches.”

Cervical Cancer Analysis Reveals New Mutations(Cancer Discov. 2017 Apr)

到目前为止，只有一种靶向治疗，已经获得FDA批准的VEGF抑制剂贝伐珠单抗(VEGF inhibitor bevacizumab)，用于治疗转移性或复发性宫颈癌患者。为了发现其他潜在的靶点，TCGA的科学家使用了多种技术来分析228个宫颈肿瘤，包括全基因组和全外显子组测序，以及对拷贝数、蛋白质水平、mRNA和miRNA表达数据以及DNA甲基化的分析。

tumor大致可以分为3类：角蛋白家族基因高或低表达的腺癌或鳞状细胞癌。角蛋白是构成生皮表皮、毛皮毛囊的主要蛋白质。
例如：two subgroups differed in expression of genes such as ARID1A, PIK3CA, and SPRR3

95%的宫颈癌是由于持续的HPV感染，该文发现一个subgroup中HPV阴性，类似子宫内膜癌。ARID1A, KRAS, PTEN高突变频率，这类少数宫颈癌可能需要另外的therapeutic options。
该研究发现了5个之前未曾报道的宫颈癌相关基因：SHKBP1, ERBB3, CASP8, HLA-A, TGFBR2。作者发现大多数肿瘤的突变似乎是由APOBEC蛋白引起的。虽然这些酶是先天免疫系统的一部分，可以改变病毒的遗传物质以抵御感染，但如果调控不当，它们也是诱变的主要来源。

Overall, “we have identified new markersunderlying cervical cancer subgroups which may translate to novel clinical therapies,” says Vellano. Additionally, he notes that CD274 and PDCD1LG2—encoding the programmed cell death ligands PD-L1 and PD-L2, respectively—were often amplified in the study tumors, suggesting a potential therapeutic role for immune checkpoint inhibitors.已经确定了子宫颈癌亚群的新标志物，这可能转化为新的临床治疗，Vellano说。此外他指出，分别编码程序性细胞死亡配体PD-L1和PD-L2的CD274和PDCD1LG2在研究肿瘤中经常扩增，这表明免疫检查点抑制剂？可能发挥治疗作用。

就像之前TCGA对子宫内膜癌和重度浆液性卵巢癌的分析一样，该研究“是对合理发展新的治疗策略的巨大参考指南”。指出，针对晚期宫颈癌的几种免疫疗法已经进入临床试验阶段，包括PD-1 blocker pembrolizumab (PD-1阻断剂pembrolizumab)(Keytruda);而CD274和PDCD1LG2扩增的发现证明了这些方法的“进一步研究”是正确的。

Whole-exome and RNA sequencing reveal novel insights into the pathogenesis of HPV associated cervical cancer[2019 Cancer Biomarkers3分]

WES identified 64 somatic mutation genes in tumors of 3 patients.
HPV阴性的宫颈癌患者比HPV阳性患者拥有更多的突变。
HPV阴性患者之间突变交集较多，HPV阴性，阳性患者之间突变交集较少。

HPV阴性患者1突变基因:CASZ1,PTPN4,WI2-2373I1.2,MUC3A,GSDMC,EMX2,AHNAK,ANO4,KRTAP4-11,KRTAP9-1,KRT15,ENTHD2,ZNF257,SSC5D,TRIOBP,IL1RAPL1,PABPC3,PROSER3
HPV阴性患者2突变基因:HNRNPCL2,C2orf16,TFAP2D,FAM185A,PABPC1,PTPRD,SECISBP2L,RASGRF1,ZNF57,PALM3,FFAR3,DMD,TRO,NAP1L3,KIAA1210,NEFH
两个阴性患者交集突变:TACR3,ADAM29,BASP1,MROH2B,HEPACAM,JPH4,TECPR2,VPS13C,SLC5A11,PRMT7,USP10,MUC16,CACNG8,TANGO2,SEZ6L,SYT2
HPV阳性突变基因: OR2L13,MUC4,ZNF141,OTOP1,TCP10,ATXN7L1,TAS2R46,SETD8,MLNR,HERC2,C2orf57,ZNF717,FADS6,SETBP1
Found 13 novel somatic mutated geneswhich were not reported by TCGA study, including SYT2, BASP1, WI2-2373I1.2, JPH4, KRTAP9- 1, HNRNPCL2, OR2L13, TAS2R46
the recurrent somatic mutated genesin cervical cancer such as PIK3CA, TTN, and FBXW7were not identifiedin this study.

TCGA研究报道的MUC4突变频率为17.2%，是宫颈癌频率最高的4个突变基因。Mucin 4，粘蛋白4是一种高度糖基化的蛋白，它是一种黏性的分离物，覆盖在气管、结肠和子宫颈等上皮表面。与正常的宫颈内细胞相比，肿瘤细胞表现出胞浆内粘蛋白的相对缺失，通常被称为粘蛋白缺失。先前的研究报道了人乳头瘤病毒相关口咽癌的MUC4错义突变和拷贝数增加。

HPV+ 相关通路研究
we can see somatic mutated genes in HPV16+ tumors specifically enriched to virus- infection related pathways including role of PKR in interferon induction and antiviral response, caveolar- mediated endocytosis signaling, and virus entry via endocytic pathways, compared with HPV-tumors.

CNV
We found similar focal amplifications in this study and TCGA result, such as in 1q21, 1q25, 3q26, and 3q28. The most significant amplification peak located in 3q26(Fig. 2A, 2B, Supplementary Fig. 1). The well-reported oncogene in 3q26 was PIK3CA, which was the most recurrent somatic mutated gene in cervi- cal cancer [14] reported by TCGA study, meanwhile. And PI3K-AKT signaling activationwas an important molecular classification of cervical cancer

EDN3was also identified as a differentially methylated genein cervical cancer by microarray analysis

Among these 127 genes, for example, PHC3amplification was reported as a possible cancer-driving genetic alteration in solid tumors by previous stud

Previous studies reported the carcinogenicity of HPV results primarily from the activity of the oncoproteins E6 and E7, which impair growth regulatory pathways

通过expression profile比对tumor和normal，发现有类似之处，故可能normal内也有部分somatic mutation。

Integrated genomic and molecular characterization of cervical cancer(2017 nature biomarker)

观察到显著的APOBEC突变模式，发现SHKBP1、ERBB3、CASP8、HLA-A和TGFBR2在宫颈癌中是新的显著突变基因。还发现免疫靶点CD274(也称PD-L1)和PDCD1LG2(也称PD-L2)以及与拉帕替尼lapatinib应答相关的BCAR4长链非编码RNA的扩增。
鉴定了一组独特的子宫内膜样宫颈癌，主要由hpv阴性肿瘤组成(高频率的KRAS、ARID1A和PTEN突变)。

HPV感染会持续存在并表达病毒致癌基因，使p53和RB失活，导致基因组不稳定性增加、体细胞突变积累。

Somatic genomic alterations

192 tumor-blood pairs.
11 tumors 归类为hypermutant，>600 mutation per sample
4 mutation per Mb(32MbX4=128)

SHKBP1, ERBB3, CASP8, HLA-A and TGFBR2as novelSMG(significantly mutated genes) in cervical cancer, and confirmed that PIK3CA, EP300, FBXW7, HLA-B, PTEN, NFE2L2, ARID1A, KRAS and MAPK1are SMGs, as previously reported
mutations in HLA-A, HLA-B, NFE2L2, MAPK1, CASP8, SHKBP1 and TGFBR2were found exclusively in squamous tumours.HLA-A、HLA-B、NFE2L2、MAPK1、CASP8、SHKBP1和TGFBR2的突变只在鳞状肿瘤中发现。
PIK3CAhad mostly activating helical-domain E542Kand E545Kmutations. PIK3CA主要有激活的螺旋结构域E542K和E545K突变
The underlying nucleotide substitution pattern in the E542K and E545K mutations is associated with mutagenesis by a subclass of APOBEC cytidine胞苷 deaminases脱氨基酶, with 150 out of 192 exomes displaying significant (q < 0.05) enrichment for the APOBEC signature。
APOBEC mutation与样本总突变数目相关，APOBEC诱变是宫颈癌主要来源之一

CNV

88 somatic copy number alterations per tumour
GISTIC2.0 analysis (with a threshold of q < 0.25) revealed 26 focal amplificationsand 37 focal deletionsalong with 23 recurrently altered whole arms
AMP:Novel recurrent focal amplification events were identified (in genomic order) at 7p11.2 (EGFR, 17%), 9p24.1 (CD274, PDCD1LG2, 21%), 13q22.1 (KLF5, 18%) and 16p13.13 (BCAR4, 20%). Other previously reported amplification events occurred at 3q26.31 (TERC, MECOM, 78%), 3q28 (TP63, 77%), 8q24.21 (MYC, PVT1, 42%), 11q22.1 (YAP1, BIRC2, BIRC3, 17%), and 17q12 (ERBB2, 17%).
DEL:Novel recurrent deletions were identified at 3p24.1 (TGFBR2, 36%) and 18q21.2 (SMAD4, 28%), in addition to previously identified deletions at 4q35.2 (FAT1, 36%) and 10q23.31 (PTEN, 31%)
鳞状肿瘤伴有11q22 (YAP1, BIRC2, BIRC3)和7p11.2 (EGFR)扩增;腺癌TGFBR2和SMAD4的缺失，ERBB2和KLF5的增加。
值得注意的是，两组均有CD274 (PD-L1)和PDCD1LG2 (PD-L2)的扩增，它们与两种关键的免疫溶细胞效应基因颗粒酶A和穿孔素18的表达显著相关(P < 0.0001)。这突出了免疫治疗策略对子宫颈癌子集的潜力。significantly (P < 0.0001) with expression of two key immune cytolytic effector genes, granzyme A and perforin18
RNA-seq analysis revealed four samples with 16p13 ZC3H7A–BCAR4 gene fusions。BCAR4是一种促进转移的长链非编码RNA，通过激活HER2/HER3通路增强抗雌激素乳腺癌的细胞增殖。拉帕替尼是一种EGFR/HER2抑制剂，在体外抑制bcar4驱动的肿瘤生长，并被认为是一种可能的治疗bcar4阳性宫颈癌的药物
KRAS, ERBB3,HLA-A,ERBB3在mRNA, methylation, miRNA and copy number variation (CNV)整合聚类中与聚类显著相关。

DNA sequencing and mutation calling

MutSig2CV6 : identify significantly mutated genes (SMGs) within the cervical cancer exome sequencing data.
All three sample subsets (all samples, squamous carcinomas only, adenocarcinomas only) without hypermutants(大于600个突变的样本不用于SMGs分析)were analysed using an FDRcut-off of 0.1. FDR values are shown in Supplementary Table 4.