Probes for INS

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.

Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.

We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway which are targets for the viral oncoproteins, E6/E7 respectively. Prospectively, BM(n=81)/BD(n=72)/OAC(n=65) FFPE specimens were subjected to IHC staining for pRb, p16INK4A , cyclin D1 , p53 and RNA in-situ hybridization (ISH) for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was also performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n=42), 18 (n=13), 6 (n=1)]. Viral load was low. Transcriptionally active HPV (DNA+ /RNA+ ) was only found in the dysplastic and adenocarcinoma group (n=21). The majority of HPV DNA+ /RNA+ BD/OAC were characterized by p16INK4Ahigh (14/21, 66.7%), pRblow (15/21, 71.4%) and p53low (20/21, 95%) and was significantly different to controls [combination of HPV DNA- /RNA- (n=94) and HPV DNA+ /RNA- cohorts (n=22)]. p53low had the strongest association with DNA+ /RNA+ oesophageal lesions (OR=23.5, 95% CI=2.94-187.8, p=0.0029). Seventeen HPV DNA+ /RNA+BD/OAC identified as p53low, were sequenced and all but one exhibited wild-type status. pRblow /p53low provided the best balance of strength of association (OR=8.0, 95% CI=2.6-25.0, p=0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA+ /RNA+ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.

Abstract

PURPOSE:

Clinical responses with programmed death (PD-1) receptor directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18 are attractive targets for therapeutic immunization, and offer an immune activation strategy that may be complementary to PD-1 inhibition.

EXPERIMENTAL DESIGN:

We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 encoding plasmids) delivered by electroporation with CELLECTRA^® constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457.

RESULTS:

MEDI0457 was associated with mild injection site reactions but no treatment related grade 3-5 adverse events (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFNg ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow cytometric analyses revealed induction of HPV16 specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%).

CONCLUSIONS:

These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.

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.