Tumour cells produce receptor activator of NF-κB ligand (RANKL) in skeletal metastases

Osteolytic bone destruction is a common complication of tumours that metastasise to bone. Several solid tumours, most notably breast carcinoma, lung carcinoma, and prostate carcinoma, commonly metastasise to bone in patients with advanced disease, where they cause osteolysis and associated pain, hypercalcemia, and fracture. It is generally accepted that osteoclasts are the only cells capable of resorbing mineralised bone. In osteolytic metastases, it has been shown that tumour cells direct osteoclastic bone resorption through a vicious cycle^1,2: in particular, tumour cell produced parathyroid hormone related protein (PTH-rP) facilitates bone resorption and, as a consequence, transforming growth factor β is released from the bone matrix and promotes the progression of bone metastases by further inducing PTH-rP production by tumour cells. Other tumour cell products, such as macrophage colony stimulating factor, interleukin 6 (IL-6), IL-11, and tumour necrosis factor α, have also been reported to be associated with tumour induced osteolysis.

However, with the identification and characterisation of a direct stimulator of osteoclastogenesis—the receptor activator of NF-kB ligand (RANKL,^3,4 also known as ODF, OPGL, and TRANCE)—a possible final common pathway for osteoclastic bone destruction has emerged. A variety of osteotropic factors, such as 1,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-6, and IL-11, have been shown to mediate osteoclast differentiation through the upregulation of RANKL expression or the downregulation of osteoprotegerin (OPG; the decoy receptor of RANKL) expression in osteoblast/stromal cells.⁵ There is also experimental evidence that tumour produced PTH-rP may stimulate osteoclastic bone resorption by enhancing RANKL expression and reducing OPG expression in the osteoblast.⁶ However, whether tumour cells directly produce RANKL, which subsequently mediates osteolysis in metastatic skeletal lesions, has not been determined.

To this end, we have investigated the expression of RANKL in the skeletal lesions of patients with carcinomas that had metastasised to bone. Sixteen cases, including breast carcinoma (four cases), lung carcinoma (six cases), prostate carcinoma (two cases), and follicular thyroid carcinoma (four cases), were collected during surgery of pathological fractures. Histological confirmation of the diagnosis in each case was based on the review of routinely prepared paraffin wax embedded tissue sections in conjunction with knowledge of the clinical and radiological findings. All patients presented with aggressive osteolytic lesions and pathological fracture, and adenocarcinoma was the predominant histological subtype (table 1). The expression of RANKL mRNA and protein was assessed using in situ hybridisation (digoxenin labelled RANKL antisense riboprobe, 0.5 ng/ml)⁷ and immunohistochemistry (mouse antihuman TRANCE monoclonal antibody from R&D, Minneapolis, Minnesota, USA; StreptABComplex/horseradish peroxidase mouse/rabbit system from Dako, Carpinteria, California, USA), respectively, on paraffin wax embedded tissue sections. Typical histological appearances of neoplastic cells in various bone metastatic tumours were revealed by haematoxylin and eosin staining (fig 1, H&E). The neoplastic cells of breast carcinoma, lung carcinoma, prostate carcinoma, and thyroid carcinoma showed strong positive hybridisation signals with RANKL riboprobes (fig 1, ISH), and also strong positive staining with anti-RANKL antibodies (fig 1, IHC). RANKL mRNA and protein were also present in osteoblasts and fibroblasts in surrounding tissues (fig 1). Table 1 summarises the percentages of tumour cells exhibiting immunoreactivity for RANKL and the intensity of immunostaining in all 16 specimens. In short, we found that both RANKL mRNA and protein were present in more than 90% and in some cases 100% of metastatic tumour cells in lesions of breast, lung, prostate, and thyroid adenocarcinoma. Therefore, we conclude that in osteolytic skeletal secondaries, metastatic tumour cells, regardless of origin, express RANKL, and may directly stimulate osteoclastic bone destruction. In support of our observations, Zhang and colleagues⁸ have recently provided evidence that tumour cells of prostate cancer are capable of inducing osteoclastogenesis in vitro, directly through the production of soluble RANKL.

Table 1.

A list of tumour bone metastases and RANKL immunoreactivity

			RANKL immunoreactivity
Case	Primary site	Diagnosis	%	Intensity
1	Breast	Adenocarcinoma	100	Intense
2	Breast	Adenocarcinoma	100	Intense
3	Breast	Adenocarcinoma	95	Intense
4	Breast	Adenocarcinoma	100	Intense
5	Lung	Adenocarcinoma	90	Intense
6	Lung	Adenocarcinoma	95	Intense
7	Lung	Adenocarcinoma	90	Intense
8	Lung	Adenocarcinoma	100	Intense
9	Lung	Adenocarcinoma	100	Intense
10	Lung	Adenocarcinoma	95	Intense
11	Prostate	Adenocarcinoma	90	Intense
12	Prostate	Adenocarcinoma	85	Faint
13	Thyroid	Follicular adenocarcinoma	90	Intense
14	Thyroid	Follicular adenocarcinoma	95	Faint to intense
15	Thyroid	Follicular adenocarcinoma	85	Faint
16	Thyroid	Follicular adenocarcinoma	100	Intense

Figure 1.

Expression of RANKL mRNA and protein in bone metastatic tumours. Signals are dark blue in colour. RANKL mRNA was present in the neoplastic cells of various metastatic tumours. Positive signals are brown in colour. RANKL protein was present at different intensities in the cytoplasm of the neoplastic cells of various metastatic tumours. All images are at ×200 magnification. In addition, when present in surrounding tissues, osteoblasts and fibroblasts also expressed RANKL mRNA and protein (indicated by arrows). H&E, haematoxylin and eosin staining; ISH, in situ hybridisation; IHC, immunohistochemistry.

Bone resorption is a necessary priming event for the establishment and propagation of tumour metastasis in bone. Our study has been conducted on the metastatic component of the primary carcinoma in the skeleton, and we did not have access to tissues of the primary site. Indeed, there is a paucity of studies that compare RANKL expression in the primary and metastatic tumours of the same patients. Brown and colleagues⁹ reported that RANKL was heterogeneously expressed in 10 of 11 prostate carcinoma specimens, and the proportion of tumour cells expressing RANKL was significantly increased in all bone metastases in comparison with non-osseous metastases or the primary prostatic tumour. Whether RANKL expression in the primary tumour is predictive of a possible propensity towards skeletal metastasis remains to be seen and could be the focus of future studies.