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A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer.

ABSTRACT: Cervical cancer, the second most common cause of cancer death in women worldwide, is significantly associated with infection of high-risk human papillomaviruses (HPVs), especially the most common genotype, HPV 16. To date, there is no established noninvasive therapy to treat cervical cancer. Methods: Here, we report a novel affitoxin that targets HPV16 E7 protein, one of the primary target proteins in molecular targeted therapy for HPV-induced cervical cancer. The affitoxin, Z_HPV16E7 affitoxin384 was generated by fusing the modified Pseudomonas Exotoxin A (PE38KDEL) to the HPV16 E7-specific affibody. The expressed and purified Z_HPV16E7 affitoxin384 was characterized using numerous methods. SPR assay, indirect immunofluorescence assay, and near-infrared (NIR) optical imaging were respectively performed to assess the targeting ability of Z_HPV16E7 affitoxin384 to HPV16 E7 protein both in vitro and in vivo. Cell viability assays and SiHa tumor-bearing nude mice were used to evaluate the efficacy of Z_{HPV16 E7} affitoxin384 in vitro and in vivo, respectively. Results: Using in vitro methods the SPR assay and indirect immunofluorescence assay showed that Z_HPV16E7 affitoxin384 targeted HPV16 E7 with high binding affinity and specificity. Significant reduction of cell viability in HPV16 positive cells was observed in the presence of Z_{HPV16 E7} affitoxin384. By NIR optical imaging, Z_{HPV16 E7} affitoxin384 specifically targeted HPV16 positive tumors in vivo. Z_HPV16E7 affitoxin384 showed significant in vivo antitumor efficacy in two kinds of tumor-bearing nude mouse models. Conclusions: Z_HPV16E7 affitoxin384 is a potent anti-cervical cancer therapeutic agent that could be effective against HPV16 positive tumors in humans.

SUBMITTER: Jiang P

PROVIDER: S-EPMC6037027 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer.

Jiang Pengfei P Wang Lude L Hou Bailong B Zhu Jinshun J Zhou Meng M Jiang Jie J Wang Ledan L Chen Shao S Zhu Shanli S Chen Jun J Zhang Lifang L

Theranostics 20180607 13

Cervical cancer, the second most common cause of cancer death in women worldwide, is significantly associated with infection of high-risk human papillomaviruses (HPVs), especially the most common genotype, HPV 16. To date, there is no established noninvasive therapy to treat cervical cancer. <b>Methods:</b> Here, we report a novel affitoxin that targets HPV16 E7 protein, one of the primary target proteins in molecular targeted therapy for HPV-induced cervical cancer. The affitoxin, Z<sub>HPV16E7 ...[more]

PMID: 30026865

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Project description:This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Project description:Simple Summary Human papillomaviruses (HPV) are one of the deadliest viruses causing cancer resulting in over 300,000 deaths annually in the world. HPV16 is the most oncogenic form of HPV and is responsible for over half of all HPV-driven tumors. The viral E7 gene encodes a powerful oncoprotein that can cause infected cells to have uncontrolled growth. A previous study showed that HPV16 isolates with amino acid alterations in the E7 gene were nearly always found in women with HPV infection but no cancer. To study the role of these variants, we expressed them in cultured cells. Most variants produced lower levels of protein, and selected variants had reduced activity in allowing cells to grow in assays that measure tumor cell invasiveness. The data indicate that inhibition of E7 could be a practical approach to treating HPV-driven cancers. Abstract The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

Dataset Information

A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer.

Publications

A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer.

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