Dose Dependent Activation of Retinoic Acid-Inducible Gene-I Promotes Both Proliferation and Apoptosis Signals in Human Head and Neck Squamous Cell Carcinoma

<div><p>The retinoic-acid-inducible gene (RIG)-like receptor (RLR) family proteins are major pathogen reorganization receptors (PRR) responsible for detection of viral RNA, which initiates antiviral response. Here, we evaluated the functional role of one RLR family member, RIG-I, in human head and neck squamous cell carcinoma (HNSCC). RIG-I is abundantly expressed both in poorly-differentiated primary cancer and lymph node metastasis, but not in normal adjacent tissues. Activation of RIG-I by transfection with low dose of 5′-triphosphate RNA (3p-RNA) induces low levels of interferon and proinflammatory cytokines and promotes NF-κB- and Akt-dependent cell proliferation, migration and invasion. In contrast, activation of RIG-I by a high dose of 3p-RNA induces robust mitochondria-derived apoptosis accompanied by decreased activation of Akt, which is independent of the interferon and TNFα receptor, but can be rescued by over-expression of constitutively active Akt. Furthermore, co-immunoprecipitation experiments indicate that the CARD domain of RIG-I is essential for inducing apoptosis by interacting with caspase-9. Together, our results reveal a dual role of RIG-I in HNSCC through regulating activation of Akt, in which RIG-I activation by low-dose viral dsRNA increases host cell surviral, whereas higher level of RIG-I activation leads to apopotosis. These findings highlight the therapeutic potential of dsRNA mediated RIG-I activation in the treatment of HNSCC.</p> </div

Apoptosis induced by transfection with high-dose 3p-RNA can be rescued by constitutively-active Akt.

<p>(A) Lysates of CAL27 cells transfected with indicated doses of 3p-RNA or BE-RNA for 16 h were immunoblotted with indicated antibodies with total Akt and β-actin as a loading control. (B) Lysates of CAL27 cells pre-transfected with HA-tagged constitutively-active Akt or empty plasmid (mock) for 24 h and then transfected with indicated doses of 3p-RNA for 16 h were immunoblotted with PARP or HA antibody with β-actin as a loading control. (C) Cell lysates from (B) were immunoprecipitated with a RIG-I antibody and immunoblotted with a caspase-9 antibody. Similar results were obtained in multiple repetitions (at least three) of these experiments.</p

Relationship between RIG-I staining intensity versus severity of HNSCC.

<p>Relationship between RIG-I staining intensity versus severity of HNSCC.</p

RIG-I is highly expressed on HNSCC cells.

<p>(A) Representative results of immunohistochemical staining of RIG-I protein (yellow) in HNSCC tissue samples. Photos were taken under 200× magnification. (B) mRNA expression of RIG-I in HNSCC tissue samples same to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058273#pone-0058273-t001" target="_blank">Table 1</a> were analyzed by Q-PCR, the lowest expression of RIG-I in normal control sample as control (*, <i>P<</i>0.001, n = 61, ANOVA). (C) CAL27 or SCC4 cells (5×10⁵ per well) were plated in 12-well plates overnight and then stimulated with PBS or LPS for 12 h or transfected with 100 ng/ml 3p-RNA for 16 h. mRNA expression levels of RIG-I and MDA-5 were analyzed by Q-PCR. The basal level of MDA-5 normalized by β-actin was used as the control. (D) CAL27 cells (2×10⁶ per well) were plated in 6-well plates and stimulated as in (C). Protein expression levels of RIG-I and MDA-5 were determined by Western blot with β-actin as the loading control.</p

Transfection of low-dose 3p-RNA induces low levels of proinflammatory cytokines and type I interferon.

<p>CAL27 cells (5×10⁵ per well) were cultured in 12-well plates overnight and then transfected with indicate doses of 3p-RNA for 16 h with BE-RNA as a control. Expression of IL-6(A), TNFα(B) and IFN-β(C) was assayed by Q-PCR. Similar results were obtained in multiple repetitions (at least three) of these experiments.</p

Transfection of high-dose 3p-RNA induces robust apoptosis.

<p>(A) CAL27 cells were transfected with the indicated dose of 3p-RNA and BE-RNA at corresponding doses as the control for 16 h and then stained with Annexin V and propidium iodide. Apoptotic cells (Annexin V positive cells) are indicated as the percentage of gated cells. (B) Lysates of CAL27 or SCC4 cells transfected with indicated doses of 3p-RNA or control for 16 h were immunoblotted with a PARP antibody. (C) Lysates of CAL27 or SCC4 cells transfected with 5 µg/ml of 3p-RNA or control for the indicated times were immunoblotted with a caspase-3 antibody. (D) Lysates of CAL27 cells transfected with 5 µg/ml of 3p-RNA or control for the indicated times were immunoblotted with a PARP antibody. (E) CAL27 cells transfected with 3p-RNA (5 µg/ml) or BE-RNA (5 µg/ml) for 4 h were stained with Rodamine 123 and propidium iodide. Apoptotic cells (R123 negative cells) are indicated as the percentage of gated cells. (F) CAL27 cells were pre-transfected with RIG-I-specific siRNA or negative control (si-Mock) 36 h before transfection with indicated doses of 3p-RNA for 16 h. Cell lysates were assayed by Western blot using a PARP antibody. β-actin was used as the loading control in (B), (C), (D), (F). Similar results were obtained in multiple repetitions (at least three) of these experiments.</p

Transfection of low-dose 3p-RNA leads to increased cell proliferation.

<p>(A) CAL27 or SCC4 cells were pre-transfected with various dose of 3p-RNA as indicated for 16 h before the MTT absorption assay with BE-RNA as a control. (B) CAL27 cells were pre-transfected with various dose of 3p-RNA (100 ng/ml) for indicated time period before the MTT absorption assay with BE-RNA as a control. (C) CAL27 cells were pre-transfected with various dose of 3p-RNA as indicated for 36 h before cell counting by FACS with BE-RNA as a control. (D) CAL27 cells treated as in A were collected for migration and invasion assays. Results are presented as fold increases over the basal level of the control. The numbers of cells in the membranes were analyzed after 24 h. (E) CAL27 cells (2×10⁶ per well) were cultured in 6-well plates and transfected with indicated doses of 3p-RNA or BE-RNA for 24 h. Cell lysates were collected and analyzed by Western blot using the indicated antibodies. (F) CAL27 cells were pre-transfected with RIG-I-specific siRNA, negative control (Si-NC) or GAPDH-specific positive control (Si-PC) for 36 h before transfection with the indicated dose of 3p-RNA for 16 h. Cell lysates were also collected for monitoring knockdown efficiency. MTT results are presented as the absorption radio. Similar results were obtained in multiple repetitions (at least three) of these experiments (A, B, C, D, F, *<i>P<</i>0.01, Student’s t-test).</p

Characteristics of surgical specimens.

<p>Note: neoadjuvant group: cisplatin-based neoadjuvant chemotherapy followed by surgery and radiotherapy. Non-neoadjuvant group: surgery followed by radiotherapy. Lower cervical metastases: cervical metastases below the plane of cricoid cartilage inferior margin.</p

Kaplan-Meier survival curves for different treatment protocols and biomarker as well as the impact of treatment procedure according to CCND1 expression.

<p>Kaplan-Meier survival curves for different treatment protocols and biomarker as well as the impact of treatment procedure according to CCND1 expression.</p

Baseline demographics for the 224 patients who participated in the study.

<p>Abbreviations: SD: standard deviation; neoadjuvant group: cisplatin-based neoadjuvant chemotherapy followed by surgery and radiotherapy group. Non-neoadjuvant group: surgery followed by radiotherapy group.</p