The genetic changes sustaining the development of cancers of unknown primary (CUP) remain elusive. The whole‐exome genomic profiling of 14 rigorously selected CUP samples did not reveal specific recurring mutation in known driver genes. However, by comparing the mutational landscape of CUPs with that of most other human tumor types, it emerged a consistent enrichment of changes in genes belonging to the axon guidance KEGG pathway. In particular, G842C mutation of PlexinB2 (PlxnB2) was predicted to be activating. Indeed, knocking down the mutated, but not the wild‐type, PlxnB2 in CUP stem cells resulted in the impairment of self‐renewal and proliferation in culture, as well as tumorigenic capacity in mice. Conversely, the genetic transfer of G842C‐PlxnB2 was sufficient to promote CUP stem cell proliferation and tumorigenesis in mice. Notably, G842C‐PlxnB2 expression in CUP cells was associated with basal EGFR phosphorylation, and EGFR blockade impaired the viability of CUP cells reliant on the mutated receptor. Moreover, the mutated PlxnB2 elicited CUP cell invasiveness, blocked by EGFR inhibitor treatment. In sum, we found that a novel activating mutation of the axon guidance gene PLXNB2 sustains proliferative autonomy and confers invasive properties to stem cells isolated from cancers of unknown primary, in EGFR‐dependent manner.

Head and neck squamous cell carcinoma (HNSCC) includes a group of aggressive malignancies characterized by the overexpression of the epidermal growth factor receptor (EGFR) in 90% of cases. Neuropilin-1 (NRP-1) acts as an EGFR co-receptor, enhancing, upon ligand stimulation, EGFR signaling in several cellular models. However, NRP-1 remains poorly characterized in HNSCC. By utilizing in vitro cellular models of HNSCC, we report that NRP-1 is involved in the regulation of EGFR signaling. In fact, NRP-1 can lead to cisplatin-induced EGFR phosphorylation, an escape mechanism activated by cancer cells upon cytotoxic stress. Furthermore, we evaluated Neuropilin-1 staining in tissue samples of an HNSCC case series (n = 218), unraveling a prognostic value for the Neuropilin-1 tissue expression. These data suggest a potential role for NRP-1 in HNSCC cancer progression, expanding the repertoire of signaling in which NRP-1 is involved and eliciting the need for further investigations on NRP-1 as a suitable target for HNSCC novel therapeutic approaches.