Receptor-interacting protein (RIP) and Sirtuin-3 (SIRT3) are on opposite sides of anoikis and tumorigenesis - PubMed (original) (raw)

. 2012 Dec 1;118(23):5800-10.

doi: 10.1002/cncr.27655. Epub 2012 Jun 6.

Affiliations

Receptor-interacting protein (RIP) and Sirtuin-3 (SIRT3) are on opposite sides of anoikis and tumorigenesis

Pachiyappan Kamarajan et al. Cancer. 2012.

Abstract

Background: Regulating cross-talk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like cancer. Previously, the authors demonstrated that anoikis activates a signaling pathway involving the CD95/Fas-mediated signaling pathway that is regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/focal adhesion kinase (FAK)-mediated survival pathways. Because it is known that sirtuin-3 (SIRT3), a nicotinamide adenine dinucleotide-dependent deacetylase, regulates cell survival, metabolism, and tumorigenesis, the authors hypothesized that SIRT3 may engage in cross-talk with Fas/RIP/integrin/FAK survival-death pathways in cancer cell systems.

Methods: Using immunohistochemical staining, immunoblotting, human tissue microarrays, and overexpression and suppression approaches in vitro and in vivo, the roles of RIP and SIRT3 were examined in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis.

Results: RIP and SIRT3 had opposite expression profiles in OSCC cells and tissues. Stable suppression of RIP enhanced SIRT3 levels, whereas stable suppression of SIRT3 did not impact RIP levels in OSCC cells. The authors observed that, as OSCC cells became anoikis-resistant, they formed multicellular aggregates or oraspheres in suspension conditions, and their expression of SIRT3 increased as their RIP expression decreased. Also, anoikis-resistant OSCC cells with higher SIRT3 and low RIP expression induced an increased tumor burden and incidence in mice, unlike their adherent OSCC cell counterparts. Furthermore, stable suppression of SIRT3 inhibited anoikis resistance and reduced tumor incidence.

Conclusions: The current results indicted that RIP is a likely upstream, negative regulator of SIRT3 in anoikis resistance, and an anoikis-resistant orasphere phenotype defined by higher SIRT3 and low RIP expression contributes to a more aggressive phenotype in OSCC development.

Copyright © 2012 American Cancer Society.

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Figures

Figure 1

Figure 1. RIP expression shows an opposite relationship to SIRT3 expression in oral squamous cell carcinoma (OSCC)

(A) RIP and SIRT3 expression in OSCC TMA specimens are illustrated. Scale bars, 200 μm for low-magnification photographs (top) and 50 μm for high-magnification photomicrographs (bottom). (B) Percentage of OSCC tissue specimens expressing RIP and SIRT3 in tongue serial sections, determined by immunohistochemical staining. Staining intensity was graded as High or Low. McNemar’s test was used to compare the two proportions and are significantly different, P ≤ .001.

Figure 2

Figure 2. RIP may be an upstream negative regulator of SIRT3

(A) Immunoblots show RIP and SIRT3 levels in normal human keratinocytes and oral squamous cell carcinoma cells (OSCC) plated for one day. β-actin served as loading control. (B) Immunoblots show RIP and SIRT3 levels after transfection with control siRNA or RIP siRNA (25 or 50 nM) in HSC-3 cells for 30 h (C) Immunoblots show SIRT3 and RIP levels after stable SIRT3 suppression using lentiviral particles (scrambled controls or SIRT3-shRNA) in UM-SCC-14A cells. (D). Immunoblots show RIP and SIRT3 levels after transfection with wild-type myc-RIP in RIP null cells (RIP−/−) for 30 h.

Figure 3

Figure 3. As OSCC cells become anoikis resistant their SIRT3 expression increases as their RIP expression decreases

(A) Phase contrast images of OSCC cells (HSC-3 and UM-SCC-14A) cultured under adherent or suspension conditions for 6 days and orasphere and single cells were separated for analysis. (B) Immunoblots showing cleaved/active caspase-3 expression, and (C) fold change of DNA fragmentation in adherent, orasphere and single cells. (D) Immunoblots showing RIP and SIRT3 expression in adherent and orasphere.

Figure 4

Figure 4. Anoikis resistant OSCC cells induce greater tumor burden in mice

(A) Images of tumor-bearing mice 6 weeks after injection with adherent or orasphere/anoikis resistant UM-SCC-14A cells. Top panels show superficial tumors and lower panels show dissected tumor. (B) Representative orasphere derived tumor section stained with H&E (left) and immnostained with antibodies for RIP (middle) and SIRT3. (C) Percentage of tissue specimens expressing RIP and SIRT3 in orasphere derived tumor sections, determined by immunohistochemical staining. Staining intensity was graded as High or Low. McNemar’s test was used to compare the two proportions and are significantly different, P ≤ .001.

Figure 5

Figure 5. SIRT3 suppression blocks orasphere formation, inhibits anoikis resistance, and reduces tumor incidence in vivo

(A) Phase contrast images of UM-SCC-14A cells transduced with scrambled shRNA (Scr-shRNA) or SIRT3 shRNA (viral transduction and puromycin selection for 10 days) then cultured under adherent or suspension (oraspheres plus single cells) conditions for 6 days. (B&C) Immunoblots show RIP and SIRT3 (B) and cleaved/active caspase-3 (C) levels in scrambled controls or SIRT3 suppressed UM-SCC-14A cells cultured under adherent or suspension (oraspheres/anoikis-resistant) conditions for 6 days. (D) Fold change in DNA fragmentation in adherent or suspension (orasphers plus single cells) conditions for 6 days. (E) Percentage of tumor incidence in nude mice injected with SIRT3 suppressed UM-SCC-14A cells (SIRT3-shRNA) or scrambled controls (Scr-shRNA) from adherent or suspension (oraspheres plus single cells) conditions after 6 weeks. Tumor incidence is shown relative to the number of animals in each group.

Figure 6

Figure 6. RIP suppression inhibits anoikis, caspase-3 activation and DNA fragmentation

(A) Immunoblots show RIP and SIRT3 levels after stable RIP suppression using lentiviral particles (scrambled controls or RIP-shRNA). (B) Phase contrast images of HSC-3 cells transduced with scrambled shRNA (Scr-shRNA) or RIP shRNA (viral transduction and puromycin selection for 10 days) then cultured under adherent or suspension (oraspheres plus single cells) conditions for 6 days. (C) Immunoblots showing cleaved/active caspase-3 expression, and (D) fold change of DNA fragmentation in adherent or suspension (orasphers plus single cells) conditions for 6 days.

Figure 7

Figure 7. Working model of anoikis resistance

Anoikis-resistant cells form multicellular aggregates (oraspheres) that express higher SIRT3 levels as their RIP expression decreases thereby promoting their survival and more aggressive phenotype in OSCC development and progression.

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