Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma

Abstract

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

Fig. 1.

Structural distribution of missense (blue) and nonsense (red) mutations in NOTCH1, organized by exon and protein domain. Below the domain graph, circles represent mutations in cSCCs, and squares represent mutations in lung SCCs. NOTCH1 driver mutations from hematologic malignancies (Catalogue of Somatic Mutations in Cancer database) are displayed above the exon graph. In these activating changes, missense mutations clustered in the NRR domain, promoting ligand-independent processing, whereas truncations in the C-terminal PEST domain enhanced protein half-life. In contrast, mutations depicted below the graph, from SCCs, appeared in EGF repeats, the juxtamembrane heterodimerization domain, and the intracellular RAM domain.

Fig. 2.

Effects and structural context of various Notch1 ectodomain mutations found in cSCC. (A) Reporter gene assay. U2OS cells lines were transfected with a Gal4-responsive luciferase reporter plasmid and with chimeric receptors that contained the indicated wild-type or mutated Notch1 extracellular domain and an intracellular Notch1/Gal4 chimera that activates transcription under the control of Gal4 binding sites. Receptors were activated by coculture with 3T3 feeder cells lacking (−) or expressing (+) the ligand Jagged2 in the absence (−) or presence (+) of a gamma secretase inhibitor (GSI). Reporter gene activity was measured after 24 h. (B) Structural context of the D469G mutation. Ribbon representation of the structure of EGF repeats 11–13 from human Notch1 (Protein Data Bank ID code 2VJ3). EGF11, blue; EGF12, green; EGF13, orange. Calcium ions are yellow; coordinating residues and interdomain contact residues are labeled and rendered as sticks; D469 is boxed. (C) Structural context of the R1594Q mutation. The Notch1 NRR is rendered in ribbon representation, with the LNRs in different shades of pink and the HD domain in either turquoise (before the S1 site), or light blue (after the S1 site). The R1594 side chain and residues within 4 Å are rendered as sticks. Right shows a magnified region of R1594 and identifies the side chains of D15343 and E1555, both of which are close enough to form charged hydrogen bonds to the R1594 side chain.

Fig. 3.

Functional characterization of the P1770S mutation. (A) Reporter gene assay. U2OS cells lines were cotransfected with a Notch-responsive luciferase reporter plasmid and plasmids encoding a constitutively processed form of the wild-type or P1770S Notch1 receptor. Reporter gene activity was measured after 24 h. (B) Electrophoretic mobility shift assay. Purified RBPJ and purified polypeptides corresponding to the RAM and ankyrin-repeat (ANK) regions of either wild-type or P1770S Notch1 were incubated in the presence of radiolabeled Hes-1 cognate DNA, as described (55).