Lethality of PAK3 and SGK2 shRNAs to human papillomavirus positive cervical cancer cells is independent of PAK3 and SGK2 knockdown

Nannan Zhou; Bo Ding; Michele Agler; Mark Cockett; Fiona McPhee

doi:10.1371/journal.pone.0117357

Lethality of PAK3 and SGK2 shRNAs to human papillomavirus positive cervical cancer cells is independent of PAK3 and SGK2 knockdown

PLoS One. 2015 Jan 23;10(1):e0117357. doi: 10.1371/journal.pone.0117357. eCollection 2015.

Authors

Nannan Zhou¹, Bo Ding¹, Michele Agler², Mark Cockett¹, Fiona McPhee¹

Affiliations

¹ Department of Virology, R&D of Bristol-Myers Squibb Company, Wallingford, Connecticut, United States of America.
² Department of Leads Discovery, R&D of Bristol-Myers Squibb Company, Wallingford, Connecticut, United States of America.

Abstract

The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Apoptosis
Cell Line, Tumor
Cell Proliferation
Female
Gene Knockdown Techniques
Genes, Lethal
HeLa Cells
Humans
Immediate-Early Proteins / antagonists & inhibitors
Immediate-Early Proteins / genetics*
Papillomavirus Infections / genetics*
Papillomavirus Infections / pathology
Protein Serine-Threonine Kinases / antagonists & inhibitors
Protein Serine-Threonine Kinases / genetics*
RNA, Small Interfering / metabolism*
Uterine Cervical Neoplasms / genetics
Uterine Cervical Neoplasms / pathology
Uterine Cervical Neoplasms / virology*
p21-Activated Kinases / antagonists & inhibitors
p21-Activated Kinases / genetics*

Substances

Immediate-Early Proteins
RNA, Small Interfering
PAK3 protein, human
Protein Serine-Threonine Kinases
p21-Activated Kinases
serum-glucocorticoid regulated kinase

Grants and funding

Funding of this study was from Bristol-Myers Squibb. BMS provided support in the form of salaries for authors [NZ, BD, MA, MC and FM], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Since Michele Agler became an employee of Boehringer Ingleheim, they have provided support in the form of a salary for her, but did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.