HPV positive neuroendocrine cervical cancer cells are dependent on Myc but not E6/E7 viral oncogenes

Hang Yuan,Faris Alkhilaiwi,Sujata Choudhary,Shuang Fang,Bassem R Haddad,Dan Zhou,David E Symer ,Naidong Wang,Xuefeng Liu,Yun Zheng ,Ewa Krawczyk,Christopher Albanese,Aleksandra Dakić ,Nancy Palechor-Ceron,Tung Wei Hou ,Maura L Gillison ,D Hartmann ,Seema Agarwal,Yukari Usuda,Danny Wangsa,Jan Blancato,Siddartha Paul,Thomas Ried,Richard Schlegel

doi:10.1038/srep45617

Abstract

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

Highlights

The viral genome was integrated into host genome adjacent to the Myc gene
We have successfully generated and propagated a culture (GUMC-395) of an HPV-16-positive, large cell neuroendocrine cervical cancer that was metastatic to the liver
GUMC-395 cells expressed the HPV-16 E6 and E7 oncogenes, but unlike all other HPV-positive cervical cancers, their proliferation was independent of these viral proteins

Summary

Introduction

RNA transcripts were detected which contained a fusion of the E7/Myc genes and, in contrast of most cervical cancers, the neuroendocrine cells harbored a p53 mutation. Since HPV is present in squamous carcinoma and adenocarcinoma of the cervix and is postulated to have a role in neuroendocrine cervical cancers, we used general HPV detection primers and HPV type-specific primers to screen the GUMC-395 cultures by PCR (Fig. 2a). Increased Myc protein in GUMC-395 was confirmed in cell extracts by western blot and in xenograft tumor sections by immunohistochemistry (Fig. 2h and i).

Results

Conclusion