Recurrent WWTR1S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix

Abstract

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)‐negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole‐exome, targeted capture, and RNA‐sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14‐3‐3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Genomic and epigenomic basis of breast invasive lobular carcinomas lacking CDH1 genetic alterations

“…Quantitative TaqMan RT-PCR for AXIN2 (Hs01063170_m1) and CDH1 (Hs01023895_m1 or Hs01023894_m1) was performed using QuantStudio3 (Applied Biosystems; ThermoFisher Scientific). Expression data were normalized to GAPDH (Hs02786624), as previously described 41 .…”

“…Phase-contrast images were obtained immediately (0 h), and 24 h and 48 h later using an EVOS XL Core Microscope (ThermoFisher Scientific). The percentage of wound closure was defined using ImageJ, as previously described 41 . Experiments were performed in triplicates in at least 3 independent experiments.…”

“…Non-viable cells were excluded and living cells were quantified using an automatic counter (Countless II, ThermoFisher, Waltham, MA, USA). Cells were reseeded in 96 well plates and cell viability was assessed 72 hours later using the CellTiter-Blue Assay (Promega), as described 41 . Absorbance was detected (560 nm excitation and 590 nm emission) using a Victor X4 Multimode Plate Reader (PerkinElmer, Hopkinton, MA, USA).…”

“…Fresh assay media was replenished every 3 days and cells were cultured for 21 days. MCF7 cells grown on 3D Matrigel culture were fixed with 4% formaldehyde (ThermoFisher Scientific) and incubated with 10% normal Goat Serum (Vector Laboratories, Burlingame, CA, USA #S-1000) 41 .…”

“…DAPI (Abcam # ab228549) was used as nuclear counterstaining. Fluorescent images were acquired using a TCS SP5 inverted confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA), as described previously 37 , 41 , 42 . No post-acquisition processing was performed, besides minor adjustments of brightness and contrast, applied equally to all images.…”