Reviewing the Osteotropism in Neuroendocrine Tumors: The Role of Epithelial-Mesenchymal Transition

Abstract

Background: Neuroendocrine tumors (NETs) metastasize to the bone. However, the incidence, clinical features, management and pathogenesis of bone involvement in NET patients have been poorly investigated. Methods: We reviewed all published reports of histologically confirmed bone metastatic NETs and explored clinical, radiological, prognostic and therapeutic characteristics in a population of 152 patients. We then evaluated immunohistochemical expression of a panel of eight epithelial-mesenchymal transition (EMT)-related factors including SNAIL, TGF-β1, CTGF, IL-11, PTHrP, EpCAM, CXCR4 and RANK in an independent cohort of 44 archival primary NETs. Biomarker expression was correlated with clinicopathological variables, including skeletal involvement, and tested for survival prediction. Results: We found that 55% of NET patients with bone metastases were male, with a median age of 55 years at diagnosis. Metastases were restricted to the skeleton in 34% of the NET population, and axial and osteoblastic lesions were prevalent. NETs differently expressed proteins involved in EMT activation. High CXCR4 (p < 0.0001) and low TGF-β1 levels (p = 0.0015) were significantly associated with increased risk of skeletal metastases, suggesting that EMT is implicated in NET osteotropism. By applying an algorithm measuring distinct immunohistochemical predictors of osteotropism on primary tumors, we were able to identify NET patients with bone metastases with a sensitivity and specificity of 91 and 100%, respectively (p < 0.0001). Patients whose primary tumors expressed CTGF (p = 0.0007) as well as the truncated form of EpCAM (p = 0.06) showed shorter survival. Conclusion: Although underestimated, bone metastases are a prominent feature of NETs, and the tumor expression of EMT markers at diagnosis may predict concurrent or subsequent skeleton colonization.

Osteotropism of neuroendocrine tumors: role of the CXCL12/CXCR4 pathway in promoting EMT in vitro

“…We have previously reported a positive nuclear staining in FFPE NET samples subjected to immunohistochemistry (IHC) using a polyclonal Ab against CXCR4 [3]. When investigated by ICC with the same Ab, all NET cell lines showed the nuclear localization of the receptor in the presence of a weak membranous labeling, as represented for CM cells in Figure 5A.…”

“…In fact, while being the only cell line with both migratory and invasive intrinsic potential, BON1 showed a profile characterized by high CXCR4 and CTGF transcription in the presence of downregulated TGF-β1 . This specific EMT signature was described in our previous work as predictive of NET bone colonization [3]. …”

“…Although NETs may develop in almost all organs, they are prevalent within the lung, the pancreas and the gastrointestinal tract, and their incidence has markedly increased over the last three decades [1, 2]. Up to 20% of patients with NETs are diagnosed with bone metastases, whose incidence and negative influence on both tumor morbidity and patient prognosis have been recently reported by our group as well as by others [3–5]. In relation to the natural history of NETs, the detection of bone involvement within 3 months from the primary NET diagnosis is apparently associated with a very dismal survival of only 12 months, and such a poor clinical outcome may be the epiphenomenon of an underlying peculiarly aggressive disease [3].…”

“…In this context, an autocrine regulatory loop between CXCL12 and CXCR4 can be hypothesized also for NETs, since evidence in other cancers suggests that persistent ligand stimulation causes CXCR4 degradation, as well as epigenetic regulation of the entire molecular synapse [7, 20, 21]. The putative association between CXCR4 overexpression, EMT activation and increased risk of NET bone metastases [3] led us to hypothesize a selection process whereby the activation of the CXCL12/CXCR4 axis skews NET cells toward a preponderance of EMT-upregulated clones. To test this hypothesis, we first ruled out the proliferation effects of CXCL12 on NET cell lines, since an accelerated proliferation could justify per se a more aggressive tumor behavior, thus promoting higher rates of skeleton colonization.…”

“…We have previously demonstrated that the epithelial-mesenchymal transition (EMT), a transdifferentiation program that promotes changes of the cell state conferring mesenchymal plasticity to epithelial cells [6], is overactive in bone-colonizing NETs [3]. In this context, overexpression of C-X-C chemokine receptor type 4 (CXCR4) in primary tumors appeared highly predictive of skeletal metastases.…”