Sonic hedgehog signaling promotes growth of oral squamous cell carcinoma cells associated with bone destruction

doi:10.1016/j.oraloncology.2011.08.026

Oral Oncology

Volume 48, Issue 1, January 2012, Pages 49-55

https://doi.org/10.1016/j.oraloncology.2011.08.026 Get rights and content

Summary

Sonic hedgehog (Shh) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of Shh in bone destruction associated with oral squamous cell carcinomas, which frequently invade the maxilla or the mandible, is still unclear. In this study we show that the use of siRNA for Shh to block SHH secreted by SAS oral squamous cell carcinoma cells suppressed the tumor growth and tumor angiogenesis of subcutaneous SAS xenografts in vivo. Moreover, blockade of Shh in SAS cells decreased tumor growth and osteoclast number in a tibial metaphysis mouse model. Significantly, we clearly show that SHH stimulated osteoclast formation in a co-culture system consisting of murine bone stromal ST2 cells and murine CD11b⁺ bone marrow cells. These findings suggest that Shh signaling is a potential target for the treatment of oral squamous cell carcinoma associated with bone destruction.

Introduction

Oral squamous cell carcinoma frequently invades the maxilla and the mandible, which action is associated with a worse prognosis.[1], [2] Bone resection as a treatment leads to the postoperative disruption of speech and swallowing function. Therefore, it is critical that a new approach be generated for the treatment of advanced oral squamous cell carcinoma.

Bone provides a favorable site for tumor growth and the aggressive behavior of breast, prostate, and oral squamous cell carcinoma tumors.[3], [4], [5] Tumor cells receive signals from the bone marrow microenvironment, which signals up-regulate the local production of growth factors, including cytokines and parathyroid hormone-related protein (PTHrP).⁶ These factors then promote the production of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblast lineage cells, and RANKL subsequently stimulates the production of bone-resorbing osteoclasts.⁷ A vicious cycle of bone destruction ensues, leading to the establishment of tumor cells in the bone marrow and resulting in increased bone resorption or osteolysis.⁸

The hedgehog family of proteins signals through the membrane receptor “Smoothened” by binding to the membrane receptor “patched”, which interaction releases Smoothened from a repressed state and allows it to signal. Hedgehog signaling is dysregulated in several cancers⁹ and has been shown to promote cancer pathogenesis.¹⁰ Recently, the hedgehog pathway was found to be associated with organ-specific metastasis to the bone.¹¹ Hedgehog transcription factor Gli2 is over-expressed in breast cancer cell lines, concomitantly leading to secretion of high levels of PTHrP; and functional over-expression of Gli2 promotes osteolytic bone metastasis by directly increasing the expression of PTHrP.¹² Recent studies revealed that Runx2 directly contributes to the osteolytic process by regulating the Indian hedgehog (IHH) -PTHrP pathway in breast cancer cells, which pathway leads to osteoclastogenesis in vivo.¹³ It is still unclear, however, as to how hedgehog signaling participates in osteoclastic bone resorption by oral squamous cell carcinoma cells. In the present study, we analyzed the effect of SHH expression on advanced oral squamous cell carcinomas and how hedgehog signaling was involved in osteoclastic bone resorption associated with the tumor invasion.

Section snippets

Cell lines and culture conditions

Human oral squamous cell carcinoma cells HSC-2, HSC-3, HSC-4, and SAS, obtained from the Human Science Research Resources Bank (Osaka, Japan); murine bone marrow stromal cells ST2, obtained from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan); and human oral squamous cell carcinoma cells UM1 and UM2¹⁴ were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). All these cell lines were characterized by genotyping conducted by

Expression of Shh mRNA in oral squamous cell carcinoma cells

To determine whether oral squamous cell carcinoma cells expressed Shh in vitro, we used RT-PCR analysis to examine UM1, UM2, SAS, HSC2, HSC3, and HSC4 cells for the expression of Shh. As shown in Fig. 1A, SAS cells highly expressed Shh, whereas the others did so only weakly. Next, to examine the role of Shh in this oral squamous cell carcinoma, we delivered separately 3 distinct (#1, #2, and #3) siRNA vectors targeting Shh into SAS cells. As shown in Fig. 1B, the expression of Shh mRNA was

Discussion

Previous studies have reported on the stimulatory effects of Shh on osteoblastogenesis and mesenchymal stem cells.[18], [19] On the other hand, hedgehog signaling has also been implicated in breast cancer-induced osteolysis.²⁰ However, the role of SHH involvement in bone destruction induced by oral squamous cell carcinomas is not well understood. Gli2 and PTHrP expression levels in oral squamous cell carcinoma cells were positively correlated, indicating that Gli2 regulates PTHrP production in

Conflict of interest

None declared.

Acknowledgements

This work was partly supported by grants from the programs Grants-in-Aid for Young Scientists (A) (to T. S.) and Scientific Research (B) (to A. S.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References (31)

D. Santini et al.
New molecular targets in bone metastases
Cancer Treat Rev
(2010)
S. Das et al.
Hedgehog signaling induced by breast cancer cells promotes osteoclastogenesis and osteolysis
J Biol Chem
(2011)
T. Nakamura et al.
Induction of osteogenic differentiation by hedgehog proteins
Biochem Biophys Res Commun
(1997)
Z. Wu et al.
RalBP1 is necessary for metastasis of human cancer cell lines
Neoplasia
(2010)
H. Nishimaki et al.
A role of activated Sonic hedgehog signaling for the cellular proliferation of oral squamous cell carcinoma cell line
Biochem Biophys Res Commun
(2004)
S. Kanda et al.
Sonic hedgehog induces capillary morphogenesis by endothelial cells through phosphoinositide 3-kinase
J Biol Chem
(2003)
J.S. Brown et al.
Patterns of invasion and routes of tumor entry into the mandible by oral squamous cell carcinoma
Head Neck
(2002)
R.J. Shaw et al.
The influence of the pattern of mandibular invasion on recurrence and survival in oral squamous cell carcinoma
Head Neck
(2004)
Y.C. Chen et al.
Breast cancer metastasis to the bone: mechanisms of bone loss
Breast Cancer Res
(2011)
A. Ebrahimi et al.
The prognostic and staging implications of bone invasion in oral squamous cell carcinoma
Cancer
(2011)

J. Sturge et al.

Bone metastasis in prostate cancer: emerging therapeutic strategies

Nat Rev Clin Oncol

(2011)

J. Liao et al.

Skeletal metastasis: Established and emerging roles of parathyroid hormone related protein (PTHrP)

Cancer Metastasis Rev

(2006)

K. Ando et al.

RANKL/RANK/OPG: key therapeutic target in bone oncology

Curr Drug Discov Technol

(2008)

M. Pasca di Magliano et al.

Hedgehog signalling in cancer formation and maintenance

Nat Rev Cancer

(2003)

S.P. Thayer et al.

Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis

Nature

(2003)

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Sonic hedgehog promotes synovial inflammation and articular damage through p38 mitogen-activated protein kinase signaling in experimental arthritis
2022, Journal of Autoimmunity
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Over the past decade, small molecule inhibitors targeting different components of SHH signaling are developed and two of the SHH inhibitors, vismodegib and sonidegib have been recommended for the treatment of locally advanced and metastatic basal cell carcinomas (BCC) [13]. Apart from the involvement in cellular proliferation and invasion of tumor, SHH signaling is reported to be correlated with bone destruction in oral squamous cell carcinomas by promoting osteoclast formation [14,15], suggesting that SHH is a potential target to prevent tumor-related bone destruction. Given the tumor-like behavior of RA FLS and its central role in synovial inflammation and joint destruction, it would be of interest to get a deeper insight into the effect of SHH signaling on RA FLS-mediated synovial inflammation and cartilage and bone damage of RA.
Activated fibroblast-like synoviocytes (FLS) play a pivotal role in synovial inflammation and joint destruction of rheumatoid arthritis (RA). The mechanisms by which sonic hedgehog (SHH) signaling promotes RA FLS-mediated chronic inflammation and tissue damage are not fully understood. The present study aims to determine the role of SHH signaling in the pathogenesis of RA and to explore the potential mechanism(s). We found that the components of SHH signaling were highly expressed in FLS and synovial tissue from patients with RA and in the joint tissue of collagen-induced arthritis (CIA) mice. Overexpression of SHH aggravated the synovial inflammation and joint destruction of CIA and exacerbated cartilage degradation in the cartilage and RA FLS-engrafted severe combined immunodeficiency (SCID) model. Conversely, inhibition of SHH signaling significantly alleviated arthritis severity and reduced cartilage destruction caused by the invasion of RA FLS in vivo. Moreover, we found that p38 mitogen-activated protein kinase (MAPK) cascade was regulated by SHH signaling in RA FLS and the level of phospho-p38 in the joint tissue of CIA was decreased after blockade of SHH signaling. Inhibition of p38 MAPK abolished the effect of SHH overexpression on synovial inflammation and articular destruction of CIA and suppressed the aggressive properties of RA FLS, which were promoted by SHH agonist. In conclusion, our study suggests that SHH signaling aggravates synovial inflammation and joint destruction of experimental arthritis and promotes the abnormal behavior of RA FLS in a p38-dependent manner. SHH-p38 MAPK signaling could be a potential target for the treatment of RA.
Exosome-derived microRNAs in oral squamous cell carcinomas impact disease prognosis
2021, Oral Oncology
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This finding is consistent with or similar to the previous reports [21,22,32]. BC6 is a novel tumor suppressor gene in medulloblastoma by repressing Sonic Hedgehog (SHH) signaling [33], and SHH signaling promotes tumor cell growth and bone destruction in OSCC [34]. Thus, BCL6/SHH axis may participate in the exosomal miR-21- and miR-155-promoted oncogenic processes of OSCC cells.
microRNA (miRNA) expression patterns have provided new insight as biomarkers of prognosis as well as novel therapeutic targets for several neoplasms. However, the role of exosomal miRNA in the prognosis of oral squamous cell carcinoma (OSCC) has not yet been completely clarified. Paired primary tumor and normal oral epithelial cells from OSCC patients were obtained, and the exosomal miRNA profiles between them were compared by miRNA microarray analysis. The miRNA levels in the serum exosomes of OSCC patients were verified by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. Finally, the biological functions and the potential as a prognostic marker of the selected miRNA candidates were analyzed in the OSCC cells and patients, respectively.
Exosomal miR-155 and miR-21 were significantly upregulated, and exosomal miR-126 was dramatically downregulated in the primary OSCC cells and the serum of OSCC patients. In the analysis of oncogenic behaviors, coculture with either miR-155-rich or miR-21-rich exosomes could promote cell proliferation and invasion accompanied with downregulation of PTEN and Bcl-6 tumor suppressors. Moreover, treatment with miR-126-rich exosomes inhibited oncogenic behaviors and oncogene EGFL7 expression in OSCC cells. Finally, exosomal miR-126 was reduced in the serum of the late-staged OSCC patients, and downregulation of blood exosomal miR-126 was associated with poor survival in OSCC patients.
Exosomal miR-155 and miR-21 are oncogenic miRNAs which suppress PTEN and Bcl-6 expression, and exosomal miR-126 acts as a tumor suppressor which downregulates EGFL7 in OSCC. Furthermore, blood exosomal miRNAs may serve as biomarkers for the diagnosis and prognosis of OSCC.
Detection of sonic hedgehog in patients undergoing orthognathic surgery
2016, International Journal of Surgery Open
Citation Excerpt :
Sonic Hedgehog (SHH) is a regulatory protein involved as a morphogen of embryonic development [2], fracture healing [3] and socket healing after tooth extraction [4]. SHH indirectly induces osteoclast formation by upregulating parathyroid hormone-related peptide (PTHrP) [5] and receptor activator for nuclear factor-κB ligand (RANKL) in osteoblasts and bone stromal cells [6]. RANKL stimulates differentiation and maturation of osteoclasts, leading to bone resorption.
Sonic Hedgehog (SHH) is a regulatory protein involved in bone fracture healing. Orthognathic surgery involves surgical osteotomy of the mandible or maxilla to restore the proper anatomic and functional position in patients with dentofacial deformity. The purpose of this study was to analyze SHH local blood serum concentrations after osteotomy to gain further understanding of the molecular regulation of the initial stage of osteotomy healing.
Serum samples (local drainage and peripheral venous) of 34 patients (24 females and 10 males, mean age was 23.4 (16–42) years) who underwent orthognathic surgery were isolated from patients at different time points during the perioperative period. The levels of SHH, soluble receptor activator of nuclear factor-κB ligand (sRANKL) and osteoprotegerin (OPG) were measured using ELISA.
SHH was detected in the local drainage immediately after osteotomy (309.5 ± 58.2 pg/ml), and decreased for 2 days after the operation (197.5 ± 43.6 pg/ml). The sRANKL local serum concentrations were at the maximum level immediately after the operation (141.4 ± 22.6 pg/ml) and decreased for 2 days (110.1 ± 23.4 pg/ml). On the other hand, the OPG concentration in the local serum was at a minimum after osteotomy (59.4 ± 4.6 pg/ml) and reached its maximum (181.5 ± 17.8 pg/ml, P < 0.01) at 2 days after osteotomy. SHH and OPG local serum levels on day 2 were associated with the amount of bleeding during the operation. The local drainage serum level of SHH of maxillary/mandibular osteotomy had a tendency to be higher than that of mandible-only osteotomy at 2 days after operation.
Elevated levels of SHH in local serum after osteotomy, especially during the initial stage of healing, indicates its importance in osteotomy healing.
Upregulation of osteoprotegerin expression correlates with bone invasion and predicts poor clinical outcome in oral cancer
2015, Oral Oncology
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In OSCC cells, RANKL, RANK and OPG expression has been detected in vitro and in vivo studies [19–22]. Recent reports have demonstrated the crucial role of the RANKL/RANK/OPG triad in osteoclast-mediated bone destruction associated with OSCCs, which frequently invade the mandible or the maxilla [23–25]. However, to date only a limited number of studies have assessed the prognostic significance of the RANKL/RANK/OPG system in oral cancer [26,27].
We aimed to determine the prognostic significance of receptor activator of nuclear factor kappa-B ligand (RANKL), RANK and osteoprotegerin (OPG) in patients with oral squamous cell carcinoma (OSCC).
The protein expression of RANKL, RANK and OPG was assessed by immunohistochemistry on pretreatment biopsies of 93 patients with locally advanced OSCC who received preoperative chemoradiotherapy (CRT). The primary endpoint was cancer-specific survival. Secondary endpoints were correlation of biomarkers with bone invasion and pathological tumor response. Kaplan–Meier curves and Cox regression models were used for survival analyses.
A significantly higher OPG expression was demonstrated in patients with malignant bone invasion and non-responders to CRT as compared to patients without bone invasion and responders (p = 0.032 and p = 0.033, respectively). Multivariate analysis revealed that higher OPG expression was independently associated with shorter cancer-specific survival (p = 0.04). The expression status of RANKL and RANK was not significantly related to clinicopathological characteristics and had no impact on survival of OSCC patients.
Upregulation of OPG expression is associated with bone invasion, poor pathological tumor regression to neoadjuvant CRT, and worse long-term cancer-specific survival in patients with locally advanced OSCC. Our results indicate that OPG may be a novel prognostic biomarker in oral cancer.
Hedgehog signaling pathway mediates tongue tumorigenesis in wild-type mice but not in Gal3-deficient mice
2014, Experimental and Molecular Pathology
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In a study using OSCC cell lines, it was shown that the activated Hh signaling pathway induced augmented 50-bromo-20-deoxyuridine (BrdU) incorporation by tumoral cells and then an increment in the cellular proliferation index (Nishimaki et al., 2004). Also, an established OSCC xenograft mouse model study revealed that nude mice transfected with Shh-inhibited cell lines presented reduced tumor growth when compared with control siRNA-transfected and parental cells (Honami et al., 2012). As a whole, these findings suggest that dysregulated Hh signaling pathway activation might be triggered during the transition from dysplasia to carcinoma in the tongue epithelial lining of Gal3 +/+ mice, probably augmenting cellular proliferation.
Oral squamous cell carcinoma (OSCC) is one of the most aggressive cancers of the oral cavity and an important cause of death worldwide. Currently, there are limited clinical tools aiding clinicians to establish its early diagnosis, and genetic and epigenetic events leading to the pathogenesis of OSCC remain unsolved. The use of carcinogen-induced knocked out mouse models would help to improve its early detection and also determine the role of proteins such as galectin-3 (Gal3) in this process. Here we used a mouse model of oral carcinogenesis employing two mouse genotypes: wild-type (Gal3 +/+) and galectin-3-deficient mice (Gal3 −/−) challenged by the carcinogen 4NQO for 16 weeks. After induction, the expression of Wnt1, Wnt3A, Shh and Gli3 proteins in tongue samples was evaluated using an immunohistochemistry approach. All samples of dysplasia and carcinoma were negative for Wnt1. Wnt3A expression was detected in both Gal3 +/+ and Gal3 −/− mice, at similar levels. Wnt3A expression did not predict tongue tumorigenesis in either genotype. Dysplastic- and carcinoma-expressing Shh was statistically significantly higher in Gal3 +/+ mice than Gal3 −/− mice (p < 0.0001), and was associated with tongue tumorigenesis only in the former. Gli3 expression decreased and increased from dysplasia to carcinoma in Gal3 +/+ and Gal3 −/− mice, respectively, although the difference was not significant. The results suggest that activated Wnt signaling is present in both mice, and that the Hh signaling pathway might play a role in tongue carcinoma development in Gal3 +/+ mice.
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Sonic hedgehog signaling promotes growth of oral squamous cell carcinoma cells associated with bone destruction

Summary

Introduction

Section snippets

Cell lines and culture conditions

Expression of Shh mRNA in oral squamous cell carcinoma cells

Discussion

Conflict of interest

Acknowledgements

Cancer Treat Rev

J Biol Chem

Biochem Biophys Res Commun

Neoplasia

Biochem Biophys Res Commun

J Biol Chem

Patterns of invasion and routes of tumor entry into the mandible by oral squamous cell carcinoma

Head Neck

The influence of the pattern of mandibular invasion on recurrence and survival in oral squamous cell carcinoma