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. Author manuscript; available in PMC: 2012 Apr 9.
Published in final edited form as: Clin Cancer Res. 2009 Dec 22;16(1):77–87. doi: 10.1158/1078-0432.CCR-08-2362

Id1 Contributes to HNSCC Survival via the NF-κB/survivin and PI3K/Akt Signaling Pathways

Jizhen Lin 1,2,*, Zhong Guan 3, Chuan Wang 1, Ling Feng 1, Yiqing Zheng 3, Emiro C Granados 1, Ellalane Bearth 1, Jie-Ren Peng 3, Patrick Gaffney 2,4, Frank G Ondrey 1,2
PMCID: PMC3321741  NIHMSID: NIHMS154609  PMID: 20028744

Abstract

Purpose

A key issue in cancer is apoptosis resistance. However, little is known about the transcription factors which contribute to cellular survival of head and neck squamous cell carcinoma (HNSCC).

Experimental design

Three batches (54, 64, and 38) of HNSCC specimens were used for cellular and molecular analyses in order to determine the major molecular signaling pathways for cellular survival in HNSCC. Animal models (cell culture and xenografts) were employed to verify the importance of apoptosis resistance in HNSCC.

Results

Inhibitor of differentiation (Id) family member, Id1, was significantly up-regulated in clinical HNSCC specimens and acted to protect keratinocytes from apoptosis. Transfection of HNSCC cells with Id1 in vitro induced the phosphorylation of Akt (p-Akt) via phosphoinositide kinase-3 (PI3K) and increased the expression of survivin via nuclear factor kappa B (NF-κB). Blockage of the both pathways by specific inhibitors (LY294002 and IκBαM, respectively) abrogated Id1-induced cell survival of keratinocytes. In vivo studies demonstrated that increased expression of Id1 allowed non-tumorigenic keratinocytes (Rhek-1A) to become tumorigenic in nude mice by increased expression of survival genes such as p-Akt and survivin. More importantly, short interfering RNA (siRNA) for Id1 significantly reduced HNSCC tumorvolume of HNSCC in xenograft studies. Analysis of clinical data verified the importance of the Id1 downstream molecule, survivin, in the prognosis of HNSCC patients.

Conclusions

The above data, taken together, suggest that Id1 and its downstream effectors are potential targets for treatment of HNSCC because of their contribution to apoptosis resistance.

Introduction

Cancer cells are by nature apoptosis resistant. Currently, the reasons for HNSCC apoptosis resistance are poorly understood t. Under normal conditions, there are two major pathways for cellular apoptosis in cancer. The first is the mitochondrial pathway characterized by intracellular caspase activation with cytochrome c participation. This is under the control of inhibitor of apoptosis proteins (IAP) which inhibit caspases (1), and is known as the intrinsic pathway. The second is the death receptor (DR) signaling pathway triggered by extracellular factors (exemplified by tumor necrosis factor alpha, TNF-α) and is, known as the extrinsic pathway.

The expression level of TNF-α is elevated in the milieu of HNSCC (2). However, cancer cells survive in such a high TNF-α enriched environment, perhaps with limited celluar apoptosis, possibly due to the NF-κB activation in HNSCC (3) which regulates IAPs (4-6) including survivin and Bcl-2 (7-9). This suggests that cellular survival plays a critical role in HNSCC through cellular apoptosis blockade.

Id1 is a transcription factor that has been identified in esophageal squamous cell carcinoma and is related to distant metastasis within 1 year of oesophagectomy (10). However, little is known about this transcription factor involved in HNSCC cellular survival. Id1 is a transcription factor which reduces cellular differentiation and increases cellular proliferation. Additionally it has been shown to be potentially upregulated in squamous cancer compared to normal skin in a very preliminary case series of squamous cancer (11). However, Id1 may also be a candidate gene for cellular survival of HNSCC, as Id1 serves as an oncogene in many tumors (12-14) and is involved in prostate cancer survival (15) and esophageal squamous cell carcinoma (10). We have recently reported that Id1 increases the proliferation of keratinocytes within 24 hours in vitro (16) and esophageal squamous cell carcinoma (10) but arrests cell growth thereafter, suggesting its involvement in cellular survival.

Recent studies indicate that Id1 is possibly linked to NF-κB in keratinocytes (16, 17) and prostate epithelial cells (7). NF-κB has been shown to increase the resistance of HNSCC cell lines to radiation (8) and improve survival of lymphoma cells in vitro (9). Therefore, it is possible that Id1, via NF-κB, increases HNSCC apoptosis resistance. Also, a recent study indicated that the expression of survivin (Baculoviral IAP repeat-containing 5, BIRC5) is under the control of NF-κB (18). Survivin, a member of the IAP family, is not detectable in normal tissues but highly upregulated in certain cancers such as adenocarcinomas of the lung, pancreas, colon, breast, and prostate (19-23). We know that survivin is also highly expressed in HNSCC (24-26) but we do not know whether survivin is under the control of Id1 via NF-κB in HNSCC.

Finally, increases in the phosphoinositide kinase-3 (PI3K)/Akt signaling pathway are also reported to aid in apoptosis resistance (27). Akt phosphorylates proapoptotic factors such as BAD and procaspase-9 as well as Forkhead transcription factor family that induces the expression of proapoptotic factors such as Fas ligands (27), making them inactive or downregulated. Whether this pathway is important for tumorigenesis of HNSCC is poorly understood at the present time.

In this study, we hypothesized that Id1 is linked to the survival of HNSCC via regulation of the NF-κB/survivin and PI3K/Akt pathways following the initial effects on cellular proliferation. Our in vivo and in vitro data demonstrated that Id1 upregulated survivin via an NF-κB dependent mechanism and simultaneously activated the Akt pathway via PI3K, contributing to keratinocyte survival. In addition, Rhek-1A, a non-tumorigenic keratinocyte cell line (28), became tumorigenic in mice after stable transfection with Id1. Additionally, inhibition of Id1 expression in HNSCC with siRNA significantly reduced xenografted tumor growth in nude mice. These findings, taken together, demonstrate the existence of an Id-1/ NFκB/survivin/AKT signaling axis in head and neck cancer apoptosis resistance.

Materials and methods

Clinical specimens

Surgical specimens (1st batch: 41 HNSCC and 13 normal tissues) were collected from the clinical patients who underwent surgery at the Department of Otolaryngology, University of Minnesota Hospital and Clinics, and used for microarray analysis, as previously published (29). This database was interrogated with respect to genes of interest in the present study. Control specimens were biopsies of normal tissues close to the cancer site. Total RNA from 54 tissue specimens was isolated using Trizol (Invitrogen, Carlsbad, CA) for microarrays. The 2nd batch of specimens (64 HNSCC and 12 vocal cord polyps) were collected from clinical patients who underwent surgery at the Department of Otolaryngology, Sun-Yat-sen University in Guangzhou of China, and used for evaluation of survivin by immunohistochemistry and clinical prognosis analysis. The 3rd batch of 50 surgical specimens (38 HNSCC and 12 normal tissues) were collected from the Department of Otolaryngology at the University of Minnesota and used for dual-labeling immunohistochemistry for Id1, NF-κB p65 subunit, p-Akt, and survivin. All specimens and clinical data in this study were procured, handled, and maintained according to the protocols approved by each Institutional Review Board (IRB).

Cell culture

Three cancer or squamous cell lines, CA9-22, Rhek-1A, and HOK16B, were used in this study. CA9-22 is a cell line established from oral cancer tissue (30, 31), HOK16B is a cell line derived from human papillomavirus (HPV) transfection of keratinocytes in the oral cavity (32), and Rhek-1A is a cell line established from the human foreskin and immortalized with SV 40 large T antigen and Kirsten sarcoma viruses (33). The CA9-22 was maintained in Gibco® RPMI 1640 (Invitrogen), the HOK-16B was maintained in keratinocyte basal media (KBM, Lon2a), and the Rhek-1A was maintained in Eagle’s minimal essential medium (MEM, Invitrogen) (34). During transient transfection of cells, Opti-MEM medium (Invitrogen,) was used (hereafter referred to as transfection medium).

Molecular reagents

The Id1 cDNA from a HNSCC specimen was cloned into a plasmid with enhanced green fluorescent protein (pEGFP, Clontech) (35) and Id1 siRNA was constructed in a similar way, as previously described (36). The p65 cDNA was cloned as previously described (37, 38). I kappa B alpha mutant (IκBαM, a kind gift of Dr. Inder Verma at Salk Institute, La Jolla, CA), a dominant negative NF-κB (39, 40), is a super repressor of NF-κB activity. Pyrrolidine dithiocarbamate (PDTC) was used as an alternative inhibitor of the NF-κB activity (41). Camptothecin, is a universal inducer of apoptosis. A specific PI3K inhibitor LY294002 was used as a specific PI3K inhibitor.

Immunohistochemistry

HNSCC and control tissues were fixed in 10% formalin, cut in a thickness of 4 microns, deparaffinized, and incubated for 90 minutes with the following primary antibodies: Id1 from Santa Cruz (1:400 dilution), NF-κB p65 subunit from Abcam (ab7970, 1:100 dilution), an “activated” form of p65 from Chemicon (MAB 3026, 1:10 dilution), and survivin from R&D Systems (1:400 dilution). Sections were then washed and incubated with FITC-, TRITC-conjugated secondary antibodies (IgG, Zymed) using the protocols as previously described (36, 42). Dual-labeled immunohistochemistry was performed, as previously described (43) to determine co-expression of Id1 and NF-κB p65 subunit, Id1 and survivin, as well as p-Akt and survivin, on clinical HNSCC specimens (batch 3) and/or mouse xenografted tumors.

Affymetrix microarrays

Affymetrix microarrays were performed as previously described (29). Briefly, cDNA, prepared from 10 μg total RNA using the double-strand DNA synthesis kit (Invitrogen), was reverse transcribed into cRNA and labeled with biotin-streptavidins using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Biotin-streptavidin labeled cRNA was then hybridized to the human U133A arrays. Expression of the Id1, Id2, Id3, Id4, PI3K, Akt1, Akt2, Akt3, NF-κB subunits, Bcl-2, and survivin genes in the HNSCC tissue specimens was identified as previously described (36). A pathway analysis tool module (PEGG) was used for evaluation of cell cycle progression and apoptosis. The results were presented as “z-scores” which weigh the entire pathway gene activity and give scores for upregulated and downregulated genes separately. Important genes involved in cellular survival were individually analyzed using t-test embedded in GeneSifter software and data is presented as individual heatmaps or merged heatmaps with P values. In addition, Affymetrix microarray analysis was performed on xenografted mouse tumors and HNSCC cell lines (CA9-22, SCC9, and NA) against their respective controls, in a similar manner.

Apoptotic assays

Nucleic acid stain was performed using Yopro-1 (Molecular Probes), which selectively passes through the plasma membranes of apoptotic cells and labels apoptotic cells green in color (44). Cells at 40% confluence were incubated with Id1 and empty vector, and apoptosis was induced by the addition of 50 μM camptothecin and stained with Yopro-1. Results are presented as % of apoptotic cells over total cells.

Trypan blue exclusion (TBE)

Briefly, cells were transfected in transfection media with Id1 and empty vector at 1.4 μg/mL for 16 hours, recovered in cell culture media for 24 hours, treated with and without PDTC at 50 μM or IκBαM at 1.4 μg/mL, and then harvested for evaluation of cell numbers after staining with Trypan blue dye. Briefly, cells were washed in PBS, and incubated in 0.3mL of 0.05% trypsin-EDTA solution for 10 minutes. Five microliters of the trypsin solution was mixed with 5 μL of Trypan blue and transferred to a hemacytometer for counting. Results are presented as viable cells (by 1×104).

Fluorescent activated cell sorting (FACS)

Cell cultures (60% confluence) were transfected in transfection media with Id1 and empty vector at 1.4 μg/mL for 16 hours, recovered in cell culture media for 24 hours, and then harvested for evaluation of antibody-stained postive cells as previously described (45). Apoptosis analysis was performed with annexin-V/7-AAD and Annexin-V-APC as instructed by the manufacturer’s maneul (BD Biosciences). Results are presented as % of viable cells (in a total of 10,000 cells counted per sample) against total cells. Effects of empty vector and Id1 on cellular survival were determined by Yopro-1, TBE, and caspase 3, or Annexin-V/7-AAD in this study. To study whether Id1-induced cellular survival occurs via PI3K, LY294002 at 50 μM was incubated with cells transfected with Id1 for 24 hours and then harvested for evaluation of cellular survival by annexin-V/7-AAD.

Luciferase assays

Cells cultured in a 12-well plate with 60% confluency were transfected with the Id1 cDNA at 1.4 μg/mL and co-transfected with NF-κB luciferase/κ-galactocidase reporters, respectively, at 1.4 μg/mL for 16 hours in the transfection medium and recovered in culture media for 24 hours. Cells were harvested for luciferase assays, as previously described (46). The activity of NF-κB luciferase over κ-galactocidase (internal control) is presented as a relative luciferase activity (RLA). The Tropix dual reporter kit (Applied Biosystems) was utilized with a Berthold Tri star flash injection luminometer.

Tumor xenograft in nude mice

Cells stably transfected with Id1 and empty vector for up to 6 months were sorted via FACSMaria cell sorter (BD Biosciences) and expanded in culture. Nude mice (6 per group) were inoculated with 1×106 cells via subcutaneous flank injection. After injection, tumor volume was measured weekly up to 10 weeks. At 10 weeks, tumors were harvested for evaluation of immunohistochemistry and global gene expression profiles by Affymetrix microarrays. To study the importance of Id1, a specific siRNA for Id1 in pSilencer 1.0-U6 (siRNA-A, 5′-aaccgcaaagtgagcaaggtgTTCAAGAGAcaccttgctcactttgcggtt-3′) was used for knocking down the Id1 gene in HNSCC cells. The siRNA control, made in our early study(36), was used. Briefly, CA9-22 cells, positive for Id1 expression, were stably transfected with siRNA construct, as above, and CA9-22 cells were then injected into 6 nude mice for tumor growth. Tumor volume was measured again, as above, from 1 to 10 weeks with control cells stably transfected with non-specific siRNA and empty vectors. For confirmation of Id1 protein inhibition in speicific and non-specific siRNA transfected xenografts, immunohistochemistry was performed using Id1 antibody and non-specific IgG as an immunohistochemistry control.

Statistical analysis

The student’s t-test was used for evaluation of differences of global gene expression between controls and experiments in vitro whereas Kaplan-Meyer survival test was used for calculation of mean and median survival time of both survivin+ and survivin− patients as well as making survival curves. Log Rank (Mantel-Cox) was then used for verification of the above data. Cox regression univariate and multivariate analyses were used for evaluation of correlates between survivin expression and clinical data (HNSCC stage, differentiation, lymph node metastasis, and distal metastasis). Fisher’s Exact Test was also used for evaluation of clinical data when the data distribution pattern is extremely uneven (such as age and distal metastasis). P values less than 0.05 were considered significant.

Results

Cell cycle progression and apoptosis are imbalanced in the HNSCC specimens

To evaluate the overall activities of cell cycle and apoptosis pathways, we used a pathway analysis parameter (z-score, a normal z-score usually between +2 and −2 as defined by GeneSifter). Normal z-scores for the both cell cycle progression and apoptosis were close to zero when 13 controls were randomly split into two groups and compared each other. Similarly, the z-scores for the both cell cycle and apoptosis within the HNSCC samples (intra-groups) were −1.59~+0.41 and −0.63~−0.15 when the 41 HNSCC specimens were randomly split into two groups and compared each other. These data indicate that z-scores for the cell cycle progression and apoptosis vary little within intra-groups, either control or cancer groups. However, the z-scores varied dramatically between inter-groups (HNSCC vs. control specimens). Among 100 genes studied on the U133A chips in the cell cycle, the expression of 34 genes was altered (>1.5-fold, with the z-score from 4.70 to −2.55, 4.70 for upregulated cell cycle genes and −2.55 for downregulated genes). Among the 78 apoptotic genes studied, the expression of 21 genes was altered in 41 HNSCC vs. 13 control (the z-score from +2.24 to −2.07, Fig. 1). It is noted that proapoptic genes were dysregulated (BIK, BAG2, and BAX upregulated but BAD down-regulated). This data suggests that many HNSCC cells go through the cell cycle and proliferate but few cells undergo apoptosis. To exclude the possibility of death receptor down-regulation, we also analyzed the extrinsic pathway on these HNSCC microarrays. It was found that genes involved in the extrinsic pathway were up-regulated in HNSCC vs. control specimens (e.g., TNF-α /Fas/TRAIL(TNF-related apoptosis-inducing ligand)/DR5/FADD (Fas-associated via death domain)/Mort1) (Fig 1). This suggests that apoptosis resistance in HNSCC cannot be attributed to the downregulation of the extrinsic pathway.

Fig. 1.

Fig. 1

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Cell cycle progression and apoptosis are imbalanced in HNSCC specimens. All gene involved in cell cycle and apoptosis were evaluated by GeneSifter using z-score as a parameter. HNSCC vs. controls had z-scores (4.70 for upregulated genes and −2.55 for downregulated genes) for the cell cycle genes but low z-scores (2.24 for upregulated and −2.07 for downregulated) for apoptosis genes (A). Dotted lines in A indicate a normal range for z-scores. Approximately 34 out of 100 genes (34%) involved in cell cycle progression had altered mRNA levels whereas only 21 out of 78 genes (26.9%) for apoptosis had altered mRNA levels (B). Microarray heatmaps demonstrated that survival-related genes were upregulated, including PI3K, Akt3, Rel B (NF-κB subunit p50), NF-κB1, NF-κB2, survivin, and Bcl-2A1 (C, survival module). Among the 34 genes altered in the cell cycle, there were 32 genes upregulated and 2 downregulated with representative ones in C (cycle module). Among the 21 apoptosis genes altered, there were 17 genes upregulated and 4 genes downregulated, with representative ones shown in C (apoptosis module). Among the Id gene family, Id1, Id2, and Id3 were upregulated but Id4 was downregulated (C, Id family). Several genes related to death receptors were upregulated in HNSCC (C, DR module). Note that some gene data is duplicated due to presence of sequence variants in gene bank. Apopt: apotosis.

Genes specific to cellular survival pathways are significantly upregulated in the HNSCC specimens

The results from this initial interrogation of the microarray data led us to postulate that cell survival related genes might be upregulated. Consequently, we then interrogated the Affymetrix microarray data for cell survival pathway-related genes. The following individual cell survival genes:Id1, Id2, PI3K, Akt3, NF-κB2, Rel B, survivin, and Bcl-2A1 were significantly upregulated in the clinical HNSCC vs. control specimens (Fig. 2A). Next, to determine a preliminary link between the above genes, Id1, p-Akt, NF-κB, and survivin, dual-labeled immunohistochemistry was performed on 50 HNSCC specimens (from batch 3). As expected, Id1 and activated NF-κB, Id1 and survivin, p-Akt and survivin, as well as p-Akt and activated NF-κB were co-expressed in the HNSCC specimens and/or xenografted tumor sections. Representative data are shown in Figure 2B-F. The normal mucosal tissues occasionally showed staining for Id1 and NF-κB but not survivin (Supplementary data 1). To examine whether Id family members (Id1-3,) are extensively expressed in HNSCC specimens, the expression of Id1-3 mRNA transcripts was examined on 41 HNSCC specimens individually. The results indicated that genes for cell survival including Id1-3, survivin, Akt 3, and NF-κB were upregulated in 58-78% of HNSCC specimens (Supplemental data 2). Id1 was found in the both cytosol and nuclei of HNSCC specimens. In batch 3 specimens, survivin expression in the cytosol was counted because of its relevance to cellular survival whereas survivin expression in the nucleus was not counted because of its non-relevance to cellular survival.

Fig. 2.

Fig. 2

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Cell survival-related genes are significantly upregulated in HNSCC specimens. Compared with control tissues, HNSCC and xenografted tumor specimens showed a significant increase in mRNA transcripts for Id1, Id2, Akt3, Rel B, survivin (A, heatmaps). Immunohistochemistry demonstrated that Id1 (dark purple) was co-expressed with NF-κB (B, intense red, green arrows, black arrows in B indicate faint p65); Id1 (red, in the nuclei) co-expressed with survivin (green, in the cytosol, C); p-Akt (red, in the cytosol) with survivin (green, in the cytosol, D); p-Akt with p65 (E, white arrowheads); and Id1 and p65 in xenografts (F, white arrowheads). NF-κB p65 subunit (activated form) was active in the edge of the xenografted mouse tumor and co-expressed with Id1 in the cellular nuclei (yellow, F). Id1 is expressed in the entire xenograft tumor whereas active p65 is expressed only in the cells which are located in the edge of xenograft tumors (F). B and F, regular microscopic graphs; C, D, and F, confocal microscopic graphs; ev: empty vector; Bar=10 μM.

Id1 increases keratinocyte survival in vitro

To study whether Id1 could be linked to cell survival in keratinocytes, we transfected Id1 into Rhek-1A cells and evaluated the keratinocytes for several cell survival markers (apoptosis as judged by caspase 3 FACSand yopro-1 stain). The efficacy of this transient transfection in Rhek-1A cells was approximately 57-70% by FACS, as previously reported (35), with an increase of Id1 mRNA by RT-PCR (data not shown). Id1 significantly reduced the apoptotic cell number of Rhek-1A as judged by Yopro-1 stain (Fig. 3A-C) and by caspase 3 FACS (Fig. 3D). Increased Id1 level was associated with an increase in the p-Akt activity by FACS (Fig. 3E). and translocation of NF-κB into the nuclei by immunohistochemistry (Fig. 3F-H). This suggests that Id1 induced cell survival via the NF-κB and p-Akt signaling pathways. To verify this, cells were transfected with Id1 in the presence and absence of NF-κB inhibitors, PDTC and IκBαM. PDTC and IκBαM abrogated the Id1-induced cell counts as judged by TBE (Fig. 3I) whereas IκBαM, a specific inhibitor of NF-κB, abrogated the Id1-induced NF-κB translocation by FACS (Fig. 3H, right). To study whether Id1-induced cellular viability is related to the PI3K pathway, cells were transfected with Id1 and then treated with PI3K inhibitor (LY294002 at 50 μM) and evaluated by 7-AAD and Annexin-V. LY294002 significantly reduced viable cells (7-AAD and Annexin-V negative cells) by Id1 (Fig. 3H, left). Similar results were found in HOK16B cells transfected with Id1 (Supplementary data 3). These data indicate that Id1 increases p-Akt and activates NF-κB which, in turn, increases apoptosis resistance, likely through an inhibition of caspase-3 activation.

Fig. 3.

Fig. 3

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Id1 is highly related to survival of Rhek-1A cells. Yopro-1 stain demonstrated that Id1 tranfected Rhek-1A cells obviously reduced the apoptotic cell numbers (B) compared with ev control (A). Statistical analysis demonstrated a significant difference of apoptotic cell numbers between ev- and Id1-transfected cells (C). Id1 significantly reduced the expression of caspase 3 by FACS (D) but significantly increased the activation of Akt (p-Akt) by FACS (E) but. Compared with those of ev (F), Id1 activated NF-κB p65 subunit by immunohistochemistry using activated p65 antibody (G, red). IκBαM, a specific inhibitor of NF-κB, abrogated the effects of Id1 on the NF-κB activation by FACS (H, left). LY294002, a specific inhibitor of the PI3K, abrogated the Id1-expressing viable keratinocytes (7-AAD and Annexin-V negative cells) by FACS (H, right) whereas PDTC and IκBαM canceled out the effect of Id1 on cellular viability by TBE (I). It is noted that transfection with ev and Id1 causes cellular apoptosis to some degree. Bar=50 μM

Id1 induces tumor growth in nude mice

To test whether Id1 expression might promote cellular survival in vivo, Id1 was stably transfected into Rhek-1A cells, Id1 expressing clones were sorted with FACSMaria and expanded in cell culture, and then injected into 5 nude mice at million cells per injection. Empty vector stably transfected Rhek-1A cells were injected into 5 nude mice as controls. Animal experiments were performed in triplicate. Id1 transfected Rhek-1A cells grew tumors in 9 out of 15 nude mice whereas empty vector transfected Rhek-1A cells grew small tumors in 3 out of 15 nude mice. The tumor volume in the Id1 groups was significantly larger than that the empty vector groups (Fig. 4A). To study whether Id1 triggers tumor growth via increased cellular survival or cell cycle progression upregulation, the global gene expression of xenograft tumors was evaluated. Id1-expressing xenografted tumors, unlike empty vector or p65 tumors, were inactive in cell cycle progression (Fig. 4B). The genes for cellular survival, similar to several HNSCC cell lines, were significantly upregulated in the xenograft Id1 tumors compared with empty vector tumors (Fig. 4C), suggesting that Id1 induced tumor growth in nude mice is primarily via increases in cellular survival.

Fig. 4.

Fig. 4

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Id1 transforms non-tumorigenic Rhek-1A cells into tumorigenic cells in nude mice. Injection of Id1 and ev transfectants at 1 million cells per flank in 15 nude mice (5 mice each group, in triplicate) demonstrated a significant difference in tumor volume between Id1- and ev-transfectants from 2 to 10 weeks (A, p<0.05). Like HNSCC cell lines (CA9-22, NA, and SCC9), xenograft tumors from Id1 vs. ev had a negative z-score for the upregulated cell cycle genes. In contrast, xenograft tumors from p65 vs. ev had a positive z-score for the upregulated cell cycle genes (B), suggesting that Id1-expressing tumors are not increasing due to active cell cycle progression, but due to cellular survival. The genes for survival were significantly higher in Id1-induced xenograft tumors than those in ev-induced xenograft tumors (C). siRNA for Id1 significantly reduced the CA9-22 xenograft tumor volume in nude mice (solid dot) compared with control (empty dot, CA9-22+ siRNA control) from 4 to 10 weeks (D). *p<0.05; surv: survivin.

Id1 siRNA inhibits tumor growth in nude mice

To test the importance of Id1 in tumor growth, one of the HNSCC cell lines, CA9-22 which express a high level of Id1 and has a z-score similar to over expressing Id1 tumor, was used for the Id1 gene knockdown with siRNA. Cancer cells with and without siRNA were injected into 5 nude mice (1 million cell flank injections). Animal experiments were performed in triplicate. CA9-22 cells transfected with Id1 siRNA grew smaller tumors than those transfected with control siRNA (Fig. 4D). Simultaneously, xenografted tumors were stained with Id1 to confirm the inhibition of Id1 protein by immunohistochemistry. Non-specific siRNA tranfected xenografts showed active Id1 protein in the nuclei whereas specific siRNA demonstrated an inhibitory effect on Id1 protein expression in the nuclei. Sections stained with non-specific IgG showed no Id1 protein signals (Supplemental data 4).

Id1 upregulates survivin via NF-κB in keratainocytes

Since survivin and Id1 were co-expressed in clinical tumors (Fig. 2), we postulated that Id1 might control the expression of survivin in HNSCC via NF-κB. To test this hypothesis, Rhek-1A cells were transfected with Id1, co-transfected with NF-κB andκ-gal reporters and harvested for evaluation of NF-κB promoter activity by luciferase assays and survivin by FACS. Id1 significantly increased NF-κB promoter activity whereas PDTC and IκBαM abrogated the Id1-induced NF-κB promoter activity (Fig. 5A). Simultaneously, Id1 significantly increased the expression of survivin and Id1-induced survivin expression was abrogated by PDTC and IκBαM as judged by FACS (Fig. 5B). This suggests that Id1 increases the expression of survivin via NF-κB. In addition, Id1 increased the translocation of NF-κB into the nuclei of Rhek-1A cells (Fig. 5D, activated p65) but PDTC abrogated the translocation of NF-κB into the nuclei of Rhek-1A cells by Id1 (Fig. 5E).

Fig. 5.

Fig. 5

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Id1 regulates the expression of survivin through NF-κB in keratinocytes. Transfection of Rhek-1A cells with Id1 significantly increased the promoter activity of NF-κB by luciferase assay and increased promoter activity was abrogated by PDTC and IκBαM (A). Id1 increased the survivin expression, but PDTC and IκBαM abrogated the Id1-induced survivin expression by FACS (B). Id1 increased the expression of survivin in the nuclei of Id1-transfected cells (D) compared with ev-transfected cells (C), but PDTC abrogated the Id1-induced survivin expression by immunohistochemistry (E) compared with Id1-transfected cells (D).

Expression of survivin in HNSCC is highly related to poor prognosis of clinical patients

To verify the importance of survivin expression as a potential factor of poor prognosis, we utilized an independent set of 64 HNSCC and 12 control specimens and stained for survivin expression (batch 3). Of the 64 clinical HNSCC specimens, 43 (67.2%) were positive for survivin in the cytosol and all the vocal cord polyps were negative for survivin by immunohistochemistry. There was a significant difference in survival time of patients between cytosol survivin+ and survivin− fractions (Fig. 6). Log Rank (Mantel-Cox) was used for verification of the above survival time data. A significant difference has seen between survivin− vs. survivin+ patients in terms of survival time (Chi-Square=6.498, P=0.011). Overall mean survival time clearly indicated that patients with survivin+ staining in the cytosol had a poor prognosis (mean survival time: 53.3 months, 95% confidence interval, 44.5–62.1 months) whereas patients with survivin− had mean survival time of 74.809 months (95% confidence interval, 65.4-84.2 months). Median survival time was analyzed, but the data was not included in this study as we could not calculate 95% confidence interval for survivin− patients, median survival has not yet been achieved in this group. The correlations between survivin and clinical data (gender, age, clinical HNSCC classification, differentiation, and metastasis) are summarized in Supplemental data 5. Association of survivin expression and clinical prognosis (Cox regression multivariate analysis) is presented in Supplemental data 6.

Fig. 6.

Fig. 6

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Patient survival is related to survivin expression in HNSCC. A, Kaplan-Meyer survival curve demonstrates a significant difference between the patients with survivin expression and those without survivin expression (Chi-Square=6.498, P = .011). The curve represents the survival status of patients at the time when the data was collected (some of the patients may be disease-free after surgery).

Discussion

Apoptosis resistance is an important mechanism for tumorigenesis. In the present study, we determined certain antiapoptotic properties of HNSCC using a global gene expression approach utilizing clinical HNSCC specimens in combination with relevant in vitro and growth characteristics in nude mice. The anti-apoptotic properties of HNSCC are attributable to the dysregulation of Id1, p-Akt, NF-κB, and survivin. This dysregulation tilts the balance of cell survival and apoptosis. Id1 contributes to this disregulation via the PI3K/Akt and NF-κB/survivin signaling pathways. The degree of change is sufficient to transform non-tumorigenic cells into tumorigenic cells, allowing growth of tumors in nude mice, a characteristic feature of survival gene expression.

Id1 induced cell survival is in part blocked by NF-κB inhibitors (IκBαM and PDTC) or PI3K inhibitor (LY294002). Interestingly, p65 alone mainly caused cell proliferation that is obviously different from Id1 in terms of tumorigenesis. If true, interesting future work in head and neck carcinogenesis examining the balance of cell survival versus proliferation may be designed around interactions between Id1 and NF-κB. In our clinical HNSCC specimens, approximately 60-75% of patients fit into this cellular survival pattern. The principal difference between xenografts and clinical specimens is that cell cycle progression was not active in Id1 xenografts compared with the clinical specimens. This difference may be attributed to genetic mutations such as tumor suppressor p53 and epigenetic changes of clinical HNSCC.

We have noted that a constitutive increase of NF-κB by p65 stable transfection in keratinocytes is critical for cell cycle progression as judged by a pathway analysis parameter (z-score). As seen in this study, p65-induced tumor (xenografted) scored 2.82. However, a study by Hinata et al demonstrated that NF-κB is involved in the maturation of human skin epithelial cells (47). Clearly, further work needs to be performed on NF κB function in normal skin epithelium and why it appears contradictory to NF-κB function in aerodigestive squamous cancer. Unlike p65, Id1 induced xenografts scored very low (−4.75) in cell cycle progression, representing a different mechanism in inducing tumor growth of nude mice characterized by cellular survival. In the current study, apoptosis is marginally increased with a z-score of 2.24 in the HNSCC specimens, perhaps attributable to the expression of a few pro-apoptotic genes such as BIK, BAX, and BAG2 against a background of a greater number of anti-apoptotic genes.

Activation of NF-κB by survival factors in normal cells usually increases the expression of Bcl-2 but not survivin. Interestingly, activation of NF-κB by Id1 leads to the expression of survivin, instead of Bcl-2, in the xenograft tumors in this study. This may explain why Id1 transfectants are tumorigenic. As seen in this study, TNF-α is expressed in clinical HNSCC specimens but has not been shown to be apoptosis promoting preclinically in head and neck cancer (48). The reason this does not lead to increased apoptosis may be attributable to IAPs. The IAP members include X-linked IAP (XIAP) and cellular IAP (cIAP). cIAP binds to TNF receptor-associated factors (TRAF1 and TRAF2), thus interfering with activation of proteases and canceling out subsequent apoptosis. NF-κB also induces TRAF1, TRAF2, c-IAP1, and c-IAP2 to suppress caspase-8 activation (5). In addition, NF-κB prevents cellular apoptosis by suppressing PTEN expression (49) through the PI3K/Akt pathway. Inhibition of NF-κB by a mutant inhibitor-κB alpha (IκBαM) attenuates resistance of human HNSCC to TNF-α caspase-mediated cell death (3). This may in part explain why increased TNFα expression in HNSCC has a limited effect on cellular apoptosis of HNSCC.

Due to the blockage of the extrinsic pathway for cellular apoptosis, HNSCC is highly dependent upon the intrinsic pathway for cell death. Unfortunately, this pathway is also inhibited. Both Id1 and NF-κB are over-expressed in HNSCC, which contributes to cellular survival. NF-κB regulates survivin (50) whereas Id1 strengthens this regulation via an increase of the NF-κB promoter activity which contributes to an increase of NF-κB constitutively. Therefore, apoptosis for HNSCC is largely blocked in the intrinsic pathway. If this is true, one should be able to construct an in vivo animal model using Id1 as a trigger. Indeed, injection of Id1-transfected keratinocytes into nude mice induced the tumor growth likely primarily due to cellular survival. Conversly, interference of Id1 expression by siRNA in HNSCC cells reduced the tumor growth in vivo probably secondary to cell survival downregualtion. However, we could not exclude the possibility that Id1 reduces the tumor volume by inhibition of angiogenesis. Our recent unpublished data indicate that Id1 contributes to angiogenesis by regulating the expression of angiogenic factors such as vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and cyclooxgenase 2 (COX2).

Id1 has recently been recognized as a clinical outcome predictor in esophageal squamous carcinoma (10). In this study, we correlated survivin with clinical outcomes of patients. As expected, survivin is highly related to the clinical prognosis of HNSCC. The reason why we exclusively focus on survivin instead of Id1 is in part because of a relatively small number of HNSCC specimens collected recently for Id1 protein analysis (batch 3) and in part because of the above study (10). We believe that focusing on the entire Id1/NF-κB/survivin signaling pathway or downstream key molecules specific for cellular survival is more relevant to clinical prognosis than an upstream molecule that has extensive effects on multiple signaling pathways. Also, survivin is uniquely expressed in cancer cells but not in normal cells whereas Id1 is mainly expressed in cancer cells but is occasionally seen in the skin basal cells and proliferating fibroblasts surrounding the tumor cells. The function of Id1 may also be offset by other helix-loop-helix transcription factors such as E-box proteins which are involved in cellular differentiation acting against Id1. In HNSCC, we have observed that some Id1 positive specimens are associated with well-differentiated cancer cells. This suggests that Id1 alone does not determine the cellular fate (proliferation or differentiation). It is the interaction between Id1 and its antagonists (helix-loop-helix transcription factors) which determine the cell fate. If this is true, Id1 predominant HNSCC may not necessarily be poorly differentiated but surely committed to cellular survival. High survivin levels in Id1+ patients may predict apoptosis resistance in HNSCC Id1+ but survivin− cells would also be expected to be apoptotically resistant, however, in this scenario, Id1’s action may be attenuated by other HLH genes that do not promote apoptosis resistance. This requires further investigation.

In summary, these data support the rationale of pharmacological inhibition of the Id1/NF-κB/survivin or Id1/PI3K/Akt pathways for HNSCC cancer therapy and suggests that inhibition of Id1 or its downstream molecule survivin removes the protection of HNSCC cells from apoptosis. Therefore, these HNSCC properties may be of significant clinical utility for head and neck cancer radiochemosensitization in order to improve long-term patient outcomes.

Supplementary Material

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Statement of relevance.

Further investigations into head and neck cancer causation will allow for the development of novel therapies designed to intervene at newly identified targets. Apoptosis resistance is very important in head and neck cancer as no therapies currently abrogate apoptosis resistance as second line treatment for failed primary disease. The current study focuses Id −1 and effector molecules that are likely associated with apoptosis resistance in this disease.. The findings of the current study suggest that Id1 and its effectors are important in the apoptosis resistance and are potential targets for therapy of head and neck cancer. The relevance is increased because the findings are reverse translated from a cohort of genomically identified poor outcome patients. Next the findings were confirmed in preclinical molecular studies, and then the concepts applied to other patient cohorts. This study suggests that Id-1 mediated signaling is an important pathway for apoptosis resistance in head and neck cancer.

Acknowledgements

Supported in part by the NIH grants (R03CA107989 and R01DC008165, To JL), NCI/NIH P30 CA77598-07 Cancer Center Support Grant (FGO)the Brainstorm Award from the University of Minnesota Cancer Center, and the Minnesota Medical Foundation. We would like to express our thanks to Robert Maisel and Bevan Yueh for their encouragements and comments, and Beverly Wuertz for her technical assistance in luciferase assays and editorial assistance in the preparation of this manuscript.

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