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Targeted Silencing of Inhibitor of Apoptosis Proteins by siRNA

Asian Pac J Cancer Prev, 14 (9), 4943-4952

Introduction

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide (Bosch et al., 2004). While earlier diagnosis and more effective treatments mean WKDW WKH SURJQRVLV RI +&& KDV VLJQLÀFDQWO\ LPSURYHG

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major obstacles to long-term survival. Although liver transplantation is considered a curative treatment for HCC, the overall recurrence rate after the procedure, attributable to distance metastasis or intrahepatic reappearance, could EHDVKLJKDVDQGUHVSHFWLYHO\:RQJ

Apoptosis is a form of programmed cell death that SOD\V D FUXFLDO ELRORJLFDO IXQFWLRQ LQ GHYHORSPHQW DQG

homeostasis in both vertebrates and invertebrates. It is also considered to be the major method of eradicating WXPRUV0DQ\UHJXODWRUVRIDSRSWRVLVKDYHEHHQLGHQWLÀHG

and one of these, inhibitor of apoptosis protein (IAP), is documented to have abnormal expression in tumor FHOOV7KH,$3JHQHHQFRGHGE\DYLUDOJHQHZDVÀUVW

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presence of a baculovirus IAP repeat (BIR) protein domain

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Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignancies, with a very poor prognosis.

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found that a major feature of tumors is a combination of unrestrained cell proliferation and impaired apoptosis.

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genes can be directed against cancers. This review will provide a brief introduction to recent developments of WKHDSSOLFDWLRQ,$3VL51$LQWXPRUVWXGLHVZLWKWKHDLPRILQVSLULQJIXWXUHWUHDWPHQWRI+&&

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Carcinoma

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domain that allows these proteins to act as E3 ubiquitin ligases (Eckelman et al., 2006). Although the main role of IAP is to act as an endogenous inhibitor of caspases WKHPDLQH[HFXWLRQHUVRIDSRSWRVLVE\ELQGLQJWRWKHLU

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irreversible cascade of events that culminates in rapid cell GHDWK&U\QVDQG<XDQ7KRUQEHUU\DQG/D]HEQLN

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three BIR domains, the third BIR mediates the binding to, and inhibition of caspase-9, an initiator caspase capable RI SURFHVVLQJ DQG WKHUHE\ DFWLYDWLQJ RWKHU FDVSDVHV

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of caspase-9 with the third BIR domain of IAPs prevents LWVKRPRGLPHUL]DWLRQLQKLELWLQJLWVDFWLYLW\6KLR]DNLHW

al., 2003). The second BIR domain and the preceding OLQNHU UHJLRQ FRRSHUDWLYHO\ PHGLDWH WKH LQWHUDFWLRQ RI

IAPs with caspase-3 and -7, the effector caspases that DUH DFWLYDWHG E\ LQLWLDWRU FDVSDVHV (IIHFWRU FDVSDVHV

are the common downstream caspase involved in the

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interaction between caspases and their cellular substrates, DQGWKXVLQKLELWVWKHSURWHRO\WLFGHJUDGDWLRQRIWKHFHOO

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group of related gene-silencing mechanisms, in which the terminal effector molecule is a short antisense RNA ,]TXLHUGR  ,W LV DOUHDG\ D YDOXDEOH DQG ZLGHO\

used, research tool for helping to understand genetic abnormalities and the molecular mechanisms of disease E\VLOHQFLQJWDUJHWHGJHQHVHVSHFLDOO\LQFDQFHUUHVHDUFK

(Chang, 2007). RNAi is a post-transcriptional gene- VLOHQFLQJWRROWKDWZRUNVE\FOHDYLQJVSHFLÀFVHTXHQFHVRI

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interfering RNAs (siRNAs). The siRNAs are bound to an 51$LQGXFHGVLOHQFLQJFRPSOH[5,6&WKURXJKZKLFK

the double-stranded siRNA is unwound (Chang, 2007).

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incorporated, guides the complex to cleave and degrade WKHSHUIHFWO\FRPSOHPHQWDU\WDUJHWP51$.DUDJLDQQLV

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Hepatocellular Carcinoma (HCC) is a major health problem, being the sixth most common cancer worldwide /ORYHW DQG %UXL[ 7KH VXUYLYDO UDWH RI SDWLHQWV

is low, due in part to aberrant tumor metastasis, UHFXUUHQFH DQG UHVLVWDQFH WR FKHPR DQG UDGLRWKHUDS\

The combination of unrestrained cell proliferation and LPSDLUHGDSRSWRVLVSOD\VDPDMRUUROHLQWKHSURJUHVVLRQ

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expression vectors, stable RNAi usage is now considered a powerful tool for silencing IAP genes in HCC research.

The following section will review the recent development RIQRYHO,$3VL51$WRROVSDUWLFXODUO\;,$36XUYLYLQ

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the initiator caspase, caspase-9, through its BIR2 and BIR3 domains, suppressing its activation and hence the activation downstream effector caspases, (Mansouri et DOFDVSDVHDQG6KL6WUXFWXUHIXQFWLRQ

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region, to bind to and inhibit caspase-3 and -7, and its third

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Increased XIAP expression has been reported in YDULHW\RIKXPDQWXPRUVLQFOXGLQJ+&&6KLUDNLHWDO

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et al., 2003). In addition, the median survival time for patients with high XIAP expression is short compared with SDWLHQWVZLWKORZRU]HUR;,$3H[SUHVVLRQ5DPSHWDO

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In the same patient population, multivariate stepwise Cox UHJUHVVLRQ DQDO\VLV VKRZHG WKDW LQ DGGLWLRQ WR WXPRU

staging (p=0.00005) and grading (p=0.00004), XIAP expression (p LVDQLQGHSHQGHQWSURJQRVWLFIDFWRU

indicating a poor prognosis in clear-cell RCC (Ramp et al., 2004).

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or cells with high XIAP expression as a negative control, ZHUHLQMHFWHGLQWRWKHÁDQNRIQXGHPLFH1HFURSV\RQ

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observations therefore suggest that altered expression of

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also associated with resistance to apoptosis, an enhanced invasiveness in vitro, which could contribute to increased PHWDVWDWLFIRFLLQYLYRDQGDVXEVHTXHQWO\UHGXFHGVXUYLYDO

time. XIAP expression could therefore be an independent prognostic factor, and novel therapeutic target for the genetic treatment of HCC patients.

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transfection, XIAP expression was down regulated at ERWKWKHP51$DQGSURWHLQOHYHOV7KH\IRXQGWKDWWKH

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different apoptosis rates after treatment with Methotrexate 07; )ORZ F\WRPHWU\ DQDO\VLV FRQILUPHG WKHVH

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and 44.6% compared with levels in the control group of

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carcinoma (Ma et al., 2009), melanoma (Hiscutt et al.,

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all clinical samples from HCC patients are XIAP-positive 6KLHWDOVRLWPD\QRWEHDQHIIHFWLYHWKHUDSHXWLF

tool for all cases of HCC.

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environmental stimuli, circulating white blood cell count, and transaminase levels were all comparable between the siRNA-treated and control mice. In contrast, the group WUHDWHGZLWKKLJKGRVH$'0GLVSOD\HGREYLRXVV\VWHPLF

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al., 2007), contains a single BIR domain, and a C-terminal 5,1*ÀQJHUGRPDLQ7ZRVSOLFHYDULDQWV/LYLQƠDQG

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expression was 3.5, compared with 27.2 months in patients ZLWKRXWLQFUHDVHG/LYLQƠp(OHYDWHG/LYLQƠ

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benign lesions of gastric mucosa, there was no detectable expression of either mRNA isoform. Their data also VKRZHGWKDW/LYLQH[SUHVVLRQZDVSRVLWLYHO\FRUUHODWHG

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control cells (p<0.05), while the cell growth index of the WUDQVIHFWHGJURXSZDVVLJQLÀFDQWO\GHFUHDVHGE\GD\V

and 4 after plating compared with the two control groups (P,QDGGLWLRQÁRZF\WRPHWULFDQDO\VLVUHYHDOHG

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through matrigel-coated membranes compared with

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with the negative control and parental cell group (p,QDGGLWLRQWKHVSRQWDQHRXVDSRSWRWLFUDWHZDV

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drugs than the negative control group. The number of DSRSWRWLFFHOOVLQGXFHGE\ÁXRURXUDFLODQGFLVSODWLQLQ

the transfected group was 2.5-3-fold elevated compared with control (p:DQJHWDO6LPLODUGDWD

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decrease the tumor volume. After palpable tumors had EHHQ FUHDWHG E\ LQMHFWLRQ RI 9LDEOH +&7 FRORQ

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decreased in the group treated with siRNA compared with the control group in a dose-dependent fashion. There were QRVLJQLÀFDQWDOWHUDWLRQVLQERG\ZHLJKWLQWKHVL51$

treated group, and no toxic reaction was detected in the NLGQH\OLYHURUEUDLQRIWKHPLFH

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consisting of three BIR domains at their N-terminii, one 5,1*GRPDLQQHDUWKH&WHUPLQXVDQGD&$5'FDVSDVH

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Bruce, also known as Appollon or BIRC6, is the ODUJHVWPHPEHURIWKH,$3IDPLO\DQGZDVÀUVWLGHQWLÀHG

in the mouse. It is a large, 530 kDa membrane-associated protein that contains a BIR domain at its N-terminus, DQGDQ(XELTXLWLQDWLRQPRWLI8EFDWLWV&WHUPLQXV

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is upregulated in certain cancers (such as gliomas) that DUHUHVLVWDQWWRVSHFLÀF'1$GDPDJLQJDJHQWVWUHDWPHQW

with antisense oligonucleotides against Bruce (Chen et al.,

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the release of caspase activators from the mitochondria 0HUU\DQG.RUVPH\HU.URHPHUDQG5HHG

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al., 2009).

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of three BIR domains at N-terminus, a central nucleotide- ELQGLQJROLJRPHUL]DWLRQGRPDLQ12'DQGD&WHUPLQDO

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proteases, caspase-3 and caspase-7, through its BIR domains (Maier et al., 2002), and associates with the initiator caspase, caspase-9 to inhibit apoptosis.

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induces the activation of the downstream effector caspase, FDVSDVH,/3WKHUHIRUHEORFNVWKHLQWULQVLFDSRSWRWLF

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documented association with malignancies, including squamous cell carcinoma, breast cancer, leukemia, colon cancer, prostate cancer, and hepatocellular carcinoma 6PLWKHWDO1RWDUEDUWRORHWDO(QGRHWDO

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et al., 2009). Endo et al. reported that NAIP is expressed LQFRORQFDQFHUDQGWKDWLWVH[SUHVVLRQLVKLJKHU\RXQJ

SDWLHQWV FRPSDUHG ZLWK WKH HOGHUO\ p<0.05) (Endo et DO.LHWDOGHWHFWHG%UXFHH[SUHVVLRQLQSDWLHQWV

with leukemia, and revealed that its over-expression was DVVRFLDWHG ZLWK UHVLVWDQFH WR FKHPRWKHUDS\ DQG SRRU

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et al., 2007).

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so understanding their independent roles in tumorigenesis requires additional studies.

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Carcinoma

Although previous reports have documented the H[SUHVVLRQ RI ,$3 IDPLO\ PHPEHUV LQFOXGLQJ ;,$3

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most studies focus on the expression of a single IAP and LWVVLJQLÀFDQFHLQ+&&7REHWWHUXQGHUVWDQGWKHIDPLO\DV

a whole, Augello et al. set out to evaluate the expression RIDOO,$3IDPLO\PHPEHUVLQKXPDQ+&&7KH\IRXQG

WKDWWKHP51$RI1$,3F,$3F,$3;,$36XUYLYLQ

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non-neoplastic liver tissue, express NAIP, accounting IRUWKLVXQH[SHFWHGREVHUYDWLRQ7KHVLJQLÀFDQFHRIWKH

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tumor grade and vascular invasion, while high c-IAP2 P51$OHYHOVDUHVLJQLÀFDQWO\FRUUHODWHGZLWKWKHDEVHQFH

of paraneoplastic capsules (p=0.02). In contrast, high NAIP mRNA levels correlate with a pseudoglandular KLVWRW\SHp=0.03), but high XIAP expression correlates ZLWK VLJQLÀFDQWO\ VKRUWHU RYHUDOO SDWLHQW VXUYLYDO 7KLV

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Conclusion and future directions

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that can render tumor cells resistant to apoptosis both LQYLYRDQGLQYLWURWKH,$3IDPLO\UHPDLQVRQHRIWKH

most promising therapeutic candidates. Moreover, cancer FHOOVWKDWRYHUH[SUHVV,$3IDPLO\PHPEHUVZLOOEHFRPH

LQFUHDVLQJO\UHVLVWDQWWRVWDQGDUGFKHPRRUUDGLRWKHUDS\

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brings new potential for cancer treatment because of its YHULÀHG DVVRFLDWLRQ ZLWK HQKDQFLQJ WKH VHQVLWLYLW\ RI

tumor cells to apoptosis, and reduced resistance to anti- WXPRUDJHQWV7KHLGHQWLÀFDWLRQRI,$3VLQKHSDWRFHOOXODU

carcinoma tissue provides the foundation of siRNA as D +&& JHQHWLF WKHUDS\ ZKLOH WKH VXEVHTXHQW VL51$

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for HCC research.

However, not all clinical samples from HCC patients test positive for a single IAP protein, and different PHPEHUVRIWKH,$3IDPLO\DFWYLDGLIIHUHQWPHFKDQLVPV

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instance, Claudia et al. reported that all measured IAPs 1$,3 F,$3 F,$3 ;,$3 6XUYLYLQ %UXFH DQG

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construct an siRNA cocktail, which is a vector containing GLIIHUHQWVL51$VIRUVSHFLÀFDOO\NQRFNLQJGRZQGLIIHUHQW

PHPEHUVRIWKH,$3IDPLO\&DOGDVHWDO

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of apoptosis in the normal pancreas, but that its role in the neoplastic pancreas depended on its sub-cellular ORFDOL]DWLRQ(VSRVLWRHWDO7KLVEULQJVFRQFHUQV

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IAPs. Although the treatment of xenograft tumors in QXGHPLFHZLWKVL51$UHYHDOHGQRREYLRXVZKROHERG\

WR[LFLW\6RQJHWDO2KHWDOWKHSRWHQWLDO

effect of IAP-siRNAs on normal tissues remains unclear.

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Acknowledgements

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apoptosis proteins (IAPs) expression and their prognostic VLJQLÀFDQFH LQ KHSDWRFHOOXODU FDUFLQRPD %0& &DQFHU

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