Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer

Abstract

PurposeProtein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells.MethodsA lentivirus-mediated short hairpin RNA (shRNA) was designed to knockdown the expression of PP4R1 in ZR-75-30 breast cancer cells. The efficiency of lentivirus-mediated shRNA infection was determined using fluorescence microscopy to observe lentivirus-mediated green fluorescent protein expression and confirmed to be over 80%. PP4R1 expression in infected ZR-75-30 cells was detected by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation and colony formation ability were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay, respectively. Flow cytometry was used to measure cell cycle progression and cell apoptosis. In addition, apoptosis makers, including poly-ADP-ribose polymerase (PARP) and caspase-3, were investigated in PP4R1-silenced ZR-75-30 cells by western blot assay.ResultsWe successfully constructed lentivirus-mediated shRNA to target PP4R1 in ZR-75-30 cells. MTT assay and colony formation assay showed the loss of PP4R1 suppressed the proliferation of ZR-75-30 cells. Flow cytometry analysis indicated cell cycle arrest and increased cell apoptosis in PP4R1 knockdown cells. Further, the apoptosis response in cells depleted of PP4R1 was illustrated by downregulation of PARP and upregulation of caspase-3.ConclusionOur results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.

Strategies for Targeting Serine/Threonine Protein Phosphatases with Small Molecules in Cancer

“…At the early stage, PPPases were simply identified as tumor suppressor proteins or oncogenic proteins. In recent years, accumulating evidence has indicated that PPPases play different roles in distinct cancers, which may be due to the diversity of regulatory subunits of PPPases. − Here, we list a few examples, mainly involving PP1, PP2A, and PP4 to illustrate that PPPases could act as both tumor promoters and tumor suppressors in specific cancers (Figure B).…”

“…For example, PR55α (a regulatory subunit) containing PP2A acts as a tumor promoter in pancreatic cancer by sustaining hyperactive tumor promoters, including YAP, AKT, ERK and Wnt; , PR61γ1 or PR61γ3 (PP2A regulatory subunit) containing PP2A acts as a tumor suppressor to stabilize p53 by dephosphorylating at either Ser37 or Thr55. , PP4 is also well-studied, and it play the opposite role in different cancers with different regulatory subunits. PPP4R1, a regulatory subunit of PP4, is overexpressed in many cancers, such as breast cancer, lung cancer, and hepatocellular carcinoma . PPP4R1 knockdown induces cancer cell proliferation inhibition by downregulating cell cycle activators .…”

“…83,84 PP4 is also well-studied, and it play the opposite role in different cancers with different regulatory subunits. PPP4R1, a regulatory subunit of PP4, is overexpressed in many cancers, such as breast cancer, 90 lung cancer, 85 and hepatocellular carcinoma. 86 PPP4R1 knockdown induces cancer cell proliferation inhibition by downregulating cell cycle activators.…”

A PP4 foszfatáz kölcsönható partnereinek azonosítása és szubsztrátum-felismerő mechanizmusának feltárása

“…A PP4 hibás működése szerepet játszhat különböző rákos megbetegedések kialakulásában, onkogénként funkcionálhat petefészekrák [87], mellrák [31,88,89],…”

“…Míg a PP4c túltermelés gátolja a proliferációt és csökkenti a mellrák sejtek metasztázisát, ezzel egyidőben növeli ezen sejtek apoptotikus aktivitástát a Bad és PEA-15 fehérjék defoszforilációján keresztül [89,95]. Ezekben a sejtekben a PP4R1 csendesítés szintén jelentősen gátolja a tumornövekedést és indukálja az apoptózist a PARP gátlásán és kaszpáz-3 aktiválása révén [88]. A PP4-nek nem csak a katalitikus és az R1, hanem az R2 alegysége is fontos a mellrák kialakulásában.…”