Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Here, we report a novel antibody drug conjugate (ADC) with the humanized anti-CD26 monoclonal antibody YS110 and triptolide (TR-1). YS110 has an inhibitory activity against the CD26-positive tumor growth via the immunological and direct pathway, such as intra-nuclear transportation of CD26 and YS110, and suppressed transcription of RNA polymerase II (Pol II) subunit POLR2A. The ADC conjugated with YS110 and an antitumor compound triptolide (TR-1), which is an inhibitor for TFIIH, one of the general transcription factors for Pol II was developed. YS110 and triptolide were crosslinked by the heterobifunctional linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and designated Y-TR1. Antitumor efficacy of Y-TR1 against malignant mesothelioma and leukemia cell lines were assessed by the in vitro cell viability assay and in vivo assay using xenografted mouse models. Y-TR1 showed significant cytotoxicity against CD26-positive cell lines but not CD26-negative counterparts in a dose-dependent manner via suppression of mRNA synthesis by impairment of the Pol II activity. The tumors in xenografted mice administered Y-TR1 was smaller than that of the unconjugated YS110 treated mice without severe toxicity. In conclusion, the novel compound Y-TR1 showed antitumor properties against CD26-positive cancer cell lines both in vitro and in vivo without toxicity. The Y-TR1 is a unique antitumor ADC and functions against Pol II.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER–plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B⁺vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER–PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR‐expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C‐terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ‐EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ‐targeting therapy for liver cancer.

Hepatocellular carcinoma (HCC) is the most common cancer with a poor prognosis. Identification of an alternative biomarker that can detect early-stage and conventional tumor marker-negative HCC is urgently needed. We found that protein kinase C delta (PKCδ) is specifically secreted from HCC cell lines into extracellular space and contributes to tumor development and that its serum levels were elevated in HCC patients. This study aimed to assess the practical usefulness of serum PKCδ for detecting HCC in chronic liver disease (CLD) patients. Serum PKCδ levels in 313 CLD patients with and without HCC (n = 187 and 126, respectively) were measured using a sandwich enzyme-linked immunosorbent assay. The diagnostic performance of PKCδ for HCC was evaluated using the receiver operating characteristic curve analysis and was compared with that of conventional markers, α-fetoprotein (AFP), and des-γ-carboxy prothrombin (DCP). Serum PKCδ levels in HCC patients were significantly higher than those in CLD patients without HCC. PKCδ distinguished HCC patients from CLD patients without HCC, with high sensitivity and specificity. Subgroup analyses revealed that the diagnostic performance of PKCδ for HCC was comparable to that of AFP and DCP, and that approximately 40% of AFP/DCP double-negative HCC patients were positive for PKCδ. PKCδ yielded better diagnostic performance for detecting solitary small-sized (ie, very early stage) HCC than AFP and DCP. There was no significant correlation between serum PKCδ and AFP/DCP levels. Serum PKCδ is a novel HCC biomarker, which is independent of and complementary to conventional markers. Specifically, PKCδ may be useful for detecting very early-stage or AFP/DCP double-negative HCC.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.