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.

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.

Protein kinase C delta (PKCδ) is a leaderless protein generally localized in the cytoplasm and nucleus; however, its extracellular unconventional protein secretion occurs exclusively in hepatocellular carcinoma (HCC) cells (but not in normal and non-cancerous hepatocytes or other gastrointestinal cancer cells) via an autophagy mechanism, despite the lack of a secretory signal. Therefore, PKCδ is detectable in the peripheral blood of HCC patients. Serum PKCδ indicates cancer-related unconventional protein secretion of an inactive form of cytosolic PKCδ and can be a unique biomarker independent of conventional markers. To examine the specificity of serum PKCδ for HCC, its levels and positivity rates were compared between 226 HCC and 108 gastrointestinal cancer patients. Furthermore, we focused on patients with malignant or benign intrahepatic tumors (17 intrahepatic cholangiocarcinoma, 42 liver metastases, and 6 focal nodular hyperplasia). A sandwich enzyme-linked immunosorbent assay was used to measure serum PKCδ levels; the optimal cutoff value was set to 57.7 ng/mL. HCC patients had significantly higher PKCδ levels and positivity rates than gastrointestinal cancer patients (median, 51.1 vs. 34.6 ng/mL; 39.8 % vs. 4.6 %; P < 0.001 for both). Thus, the specificity was 95.4 %. Focusing on intrahepatic tumors, 2 (4.8 %) of 42 gastrointestinal cancer patients with liver metastasis were positive for PKCδ. Notably, all patients with intrahepatic cholangiocarcinoma and focal nodular hyperplasia were negative for PKCδ, irrespective of their intrahepatic tumors being malignant or benign. Serum PKCδ is an extracellularly secreted protein specific for HCC that can be a novel diagnostic biomarker because it distinguishes HCC from other gastrointestinal cancers and intrahepatic non-HCC tumors.

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