Dying tumor cells stimulate proliferation of living tumor cells via caspase‐dependent protein kinase Cδ activation in pancreatic ductal adenocarcinoma

Abstract

Pancreatic cancer is one of the most lethal human cancers, and radiotherapy is often implemented for locally advanced pancreatic ductal adenocarcinoma. Tumor cell repopulation is a major challenge in treating cancers after radiotherapy. In order to address the problem of tumor repopulation, our previous studies have demonstrated that dying cells stimulate the proliferation of living tumor cells after radiotherapy. In particular, dying cells undergoing apoptosis also activate survival or proliferation signals and release growth factors to surrounding living cells. In the present study, we used an in vitro model to examine the possible mechanisms for dying cell-stimulated tumor repopulation in pancreatic cancer. In this model, a small number of living, luciferase-labeled pancreatic cancer cells (reporter) were seeded onto a layer of a much larger number of irradiated, unlabeled pancreatic cancer cells and the growth of the living cells was measured over time as a gauge of tumor repopulation. Our results indicate that irradiated, dying Panc1 feeder cells significantly stimulated the proliferation of living Panc1 reporter cells. Importantly, we identified that the percentage of apoptotic cells and the cleavage of caspases 3 and 7 and protein kinase Cδ (PKCδ) were increased in irradiated Panc1 cells. We presumed that caspases 3 and 7 and PKCδ as integral mediators in the process of dying pancreatic cancer cell stimulation of living tumor cell growth. In order to demonstrate the importance of caspases 3, 7 and PKCδ, we introduced dominant-negative mutants of caspase 3 (DN_C3), caspase 7 (DN_C7), or PKCδ (DN_PKCδ) into Panc1 cells using lentiviral vectors. The stably transduced Panc1 cells were irradiated and used as feeders and we found a significant decrease in the growth of living Panc1 reporter cells when compared with irradiated wild-type Panc1 cells as feeders. Moreover, the role of PKCδ in the growth stimulation of living tumor cells was further confirmed using a pan PKC inhibitor GF109203× and a specific PKCδ inhibitor, rottlerin. Additionally, we found significantly increased phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK1/2) in the irradiated Panc1 cells. Mechanistically, PKCδ cleavage was attenuated in both DN_C3 and DN_C7 transduced Panc1 cells, and both Akt and p38 MAPK phosphorylation were attenuated in DN_PKCδ transduced Panc1 cells following radiation. Thus, this report suggests a novel finding that cellular signaling from caspase 3/7-PKCδ-Akt/p38 MAPK is crucial to the repopulation in Panc1 cells after radiotherapy.

Targeting protein kinase C subtypes in pancreatic cancer

“…Dying pancreatic cancer cells after radiotherapy can act as feeder cells for living tumor cells. It was shown that this is mediated by a Caspase 3/PKCδ signaling pathway that is active in dying tumor cells [21]. …”

The Sound of Silence

“…These findings were validated in breast cancer, melanoma, and pancreatic ductal adenocarcinoma cell lines (Cheng et al, 2015; Donato et al, 2014; Kurtova et al, 2015). …”

“…In addition to the Caspase-3/iPLA 2 /PGE 2 signaling, in certain cancer cell lines such as the Panc1 line derived from ductal carcinoma, there is also a role for 7-PKCδ-Akt/p38 MAPK-stimulated mitogen production (Cheng et al, 2015). Also, there is evidence of Sonic Hedgehog modulating AiP, feeding back on dying cells in a paracrine manner to regulate WNT signaling via SFRP1 (secreted frizzled related protein 1).…”

Protein kinase C isoforms in the normal pancreas and in pancreatic disease

“…PKCδ has been shown to drive anchorage-independent growth through activation of the PI3K-Akt pathway, and treatment of Panc1 cells overexpressing PKCδ with the PI3K inhibitor LY294002 prevented soft agar colony formation [64]. Besides its roles in cell survival, anchorage independent growth and proliferation, PKCδ is also implicated in cell metastasis [82], and overexpression of PKCδ enhances the metastatic capability of Panc1 cells in xenografts [64]. …”

“…Ionizing radiation causes cleavage and activation of caspases 3 and 7, of which caspase 3 cleaves PKCδ, resulting in an active, unregulated kinase fragment [95, 96]. This active PKCδ fragment then activates Akt to drive tumor cell proliferation [82]. While PKCε can be activated by caspase 3 by a similar mechanism, it so far has not been implicated in this pathway [96].…”