Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer.
Significance:
PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.
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.
<div>Abstract<p>Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi–independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer–related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer.</p>Significance:<p>PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.</p></div>
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