The role of PKD2 in HSP90,- hypoxia- and TGF beta-mediated epithelial-to-mesenchymal transition and metastasis
Final Report Abstract
The PKD family belongs to the calcium/calmodulin-dependent protein kinase superfamily and comprises PKD1/PKCgamma, PKD2 and PKD3/PKCv. We have recently shown that Protein kinase D2 (PKD2) is a crucial regulator of tumour cell - endothelial cell communication in gastrointestinal tumors. Our findings indicated that PKD2 contributes to blood vessel formation in hypoxic tumours by regulating hypoxia-inducing factor (HIF) 1alpha, a master oxygen sensor protein. Furthermore, we identified PKD2 as a novel client of the heat shock protein (HSP) 90, a molecular chaperone involved in the correct folding of proteins, and demonstrated that the kinase participates to the tumor angiogenic program orchestrated by chaperone. Hypoxia and HSP90 were reported to be involved also in another aspect of tumorigenesis, namely, epithelial-to-mesenchymal transition (EMT), a multi-step process in which cancer cells acquire a migratory phenotype and invasive capacity. Our findings reveal an essential role of PKD2 in promoting EMT. Thus, ectopic PKD2 was paralleled by loss of epithelial markers (e.g. E-cadherin), gain of mesenchymal morphology and mesenchymal markers (e.g. vimentin) as well as elevated levels of several EMT-specific transcription factors (e.g. ZEB1, ZEB2). By contrast, PKD2 abrogation was associated with EMT reversal in hypoxic cancer cells. Moreover, PKD2 participated to the EMT program coordinated by HSP90. Disruption of the mesenchymal structures and the loss of vimentin following pharmacologic HSP90 inhibition were restored upon over-expression of PKD2 both in vitro and in in vivo tumour xenograft. In addition, our experiments indicate that secretion of TGFß is promoted by PKD2. Consistently, abrogation of kinase was associated with impaired hypoxia-induced TGFß secretion indicating an interference e of PKD2 with TGFß-induced EMT in hypoxic tumours. We previously showed that TGFß activates the NF-KB transcription factors. Here we could demonstrate that TGFß induced NF-KB activation is negatively-impacted upon PKD2 depletion indicative of a TGFß->PKD2->NF-KB activation axis during EMT. During the second financing period we also invested efforts in characterizing the contribution of HSP90/PKD2 to new potential therapy avenues by involving cold physical plasma, an emerging and less aggressive tumour therapy, associated with reactive oxygen species (ROS) generation. Our experiments indicate that HSP90 cleavage by plasma-triggered ROS was associated with PKD2 degradation. Most importantly, pre-sensitization of cancer cells with 50 nM PU-H71 HSP90 inhibitor was sufficient to significantly boost the cell death triggered by 30 sec cold-plasma therapy. Given the long development times, rising costs and outcome often unsatisfying of various anti-cancer drugs, we joined the effort in investigating the repurposing of existing medications already on the market with a high safety record and cost efficacy. Sildenafil is a phosphodiesterase 5 (PDE5)-inhibiting drug that has been reported to trigger tumour cell killing by altering the expression of several chaperones, including HSP90. Administration of sildenafil was associated not only with impaired proliferation in several cancer cell lines in vitro and a significant decrease of tumour size in vivo but also by a decreased HSP90 expression and substantial degradation of PKD2. We also investigated the contribution to cancer of other protein kinases such as pyruvate kinase M2 (PKM2) initially reported as a crucial modulator of glycolysis. Indeed, examination of PKM2 expression by immunohistochemistry in a set of 34 human primary pancreatic ductal adenocarcinomas or metastases to liver, soft tissue, lymph node and lung revealed moderate to strong PKM2 immuno-reactivity. Our experiments showed that PKM2 abrogation correlated with a marked reduction in cell proliferation in vitro, as well as smaller tumours and impaired vascularization in vivo. Mechanistically, our data favour a scenario where the hypoxic insult is followed by translocation to nucleus and interaction of PKM2 and NF-KB/p65 which culminates with VEGF secretion and a boost in blood vessel formation that in turn contributes to tumour growth. Altogether, our findings delineate once again the essential role played by protein kinases in various cancer hallmarks.
Publications
- HSP90 supports tumor growth and angiogenesis through PRKD2 protein stabilization. Cancer Research (2014), 74(23):7125-7136
Ninel Azoitei, Kristina Diepold, Cornelia Brunner, Arefeh Rouhi, Felicitas Genze, Alexander Becher, Hans Kestler, Johan van Lint, Gabriela Chiosis, John Koren III, Stefan Fröhling, Claudia Scholl, Thomas Seufferlein
(See online at https://doi.org/10.1158/0008-5472.CAN-14-1017) - PKD2: A two-pronged kinase crucial for the tumor-supporting activity of HSP90. Molecular & Cellular Oncology, Vol. 2. 2015, Issue 2, e981444.
Azoitei N., Fröhling S., Scholl C., Seufferlein T.
(See online at https://doi.org/10.4161/23723556.2014.981444) - PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation. Molecular Cancer, (2016), Jan 6;15(1):3
Azoitei N, Becher A, Steinestel K, Rouhi A, Diepold K, Genze F, Simmet T, Seufferlein T
(See online at https://doi.org/10.1186/s12943-015-0490) - STK33 participates to HSP90-supported angiogenic program in hypoxic tumors by regulating HIF-1α/VEGF signalling pathway. Oncotarget (2017), 8(44):77474-77488
Liu Y, Steinestel K, Rouhi A, Armacki M, Diepold K, Chiosis G, Simmet T, Seufferlein T, Azoitei N
(See online at https://doi.org/10.18632/oncotarget.20535) - Protein kinase D2: a versatile player in cancer biology. Oncogene (2018), 37(10):1263-1278
Azoitei N, Cobbaut M, Becher A, Van Lint J , Seufferlein T
(See online at https://doi.org/10.1038/s41388-017-0052-8) - Physical plasma-triggered ROS induces tumor cell death upon cleavage of HSP90 chaperone. Scientific Reports (2019), Mar 11;9(1):4112
Bekeschus S, Lippert M, Diepold K, Chiosis G , Seufferlein T, Azoitei N
(See online at https://doi.org/10.1038/s41598-019-38580-0) - Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2. Carcinogenesis, Vol. 41. 2020, Issue 10, pp. 1421–1431.
Chen L., Liu Y., Becher A., Diepold K., Schmid E., Fehn A., Brunner C., Rouhi A., Chiosis G., Cronauer M., Seufferlein T., Azoitei N.
(See online at https://doi.org/10.1093/carcin/bgaa001)
