Probes for INS

Abstract

Inturned (INTU), a cilia and planar polarity effector, performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for intraflagellar transport A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily responds to the Hh pathway activation. Depleting Intu blocked Smo agonist-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.

Abstract

PURPOSE:

Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double strand break (DSB) repair and high sensitivity to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In the present study we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancers (BrC) tissue: the RECAP test.

METHODS:

Fresh samples of 170 primary BrC were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor infiltrating lymphocytes (TILs) and microsatellite instability (MSI).

RESULTS:

RECAP was completed successfully in 148 out of 170 samples (87%). 24 tumors showed HRD (16%), while 6 tumors were HR intermediate (HRi) (4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas 7 HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts (p=0.023) compared to HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in BrC (1%) (p=0.017).

CONCLUSION:

RECAP is a robust functional HR assay detecting both BRCA1/2 deficient and BRCA1/2 proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh+/Notch+ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh+++/Notch− basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap-null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK-JUN signaling, a well-established tumor-driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug-resistant BCC.