Deciphering the Role of Protein Kinase D1 (PKD1) in Cellular Proliferation

Author Manuscript Published OnlineFirst on July 16, 2019; DOI: 10.1158/1541-7786.MCR-19-0125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Deciphering the role of protein kinase D1 (PKD1) in cellular proliferation

2 Ilige Youssefa,b and Jean-Marc Ricorta,b,c,*

4 aCentre National de la Recherche Scientifique, CNRS UMR_8113, Laboratoire de Biologie et

5 Pharmacologie Appliquée, F-94230 Cachan, France

6 bÉcole Normale Supérieure Paris-Saclay, Université Paris-Saclay, F-94230 Cachan, France

7 cCentre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes,

8 Université Paris Diderot, F-75006 Paris, France

10 Corresponding Author: Ricort Jean-Marc; email: [email protected]; address: Centre

11 de Recherche des Cordeliers, Laboratoire de Physiopathologie Orale Moléculaire, 15 rue de l’école de

12 médecine, 75006 Paris, France

14 Running Title: The role of PKD1 in cell proliferation

16 Disclosure of Potential Conflicts of Interest

17 No potential conflicts of interest were disclosed.

Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 16, 2019; DOI: 10.1158/1541-7786.MCR-19-0125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

26 Abstract

27 Protein kinase D1 (PKD1) is a serine/threonine kinase that belongs to the calcium/calmodulin-

28 dependent kinase family, and is involved in multiple mechanisms implicated in tumor

29 progression such as cell motility, invasion, proliferation, protein transport, and apoptosis. While

30 it is expressed in most tissues in the normal state, PKD1 expression may increase or decrease

31 during tumorigenesis, and its role in proliferation is context-dependent and poorly understood. In

32 this review, we present and discuss the current landscape of studies investigating the role of

33 PKD1 in the proliferation of both cancerous and normal cells. Indeed, as a potential therapeutic

34 target, deciphering whether PKD1 exerts a pro- or anti-proliferative effect, and under what

35 conditions, is of paramount importance.

36 Introduction

38 PKD1, also called PKCµ, is a serine/threonine kinase that belongs to the PKD family, a

39 subgroup of the calcium/calmodulin-dependent kinase (CAMK) family (1). PKD1 is a 912

40 amino acid residue protein with an apparent molecular weight of 115 kDa which contains a

41 carboxy-terminus catalytic domain and a regulatory domain at the amino-terminus. The latter

42 regulates the catalytic activity of PKD1 by maintaining the protein in an inactive state through an

43 auto-inhibitory mechanism exerted towards the catalytic domain (2). PKD1 can be activated by a

44 wide variety of extracellular stimuli including growth factors, vasoactive peptides, chemokines,

45 neuropeptides, phorbol esters, and others. To date, the best characterized signalling pathway

46 responsible for the activation of PKD1 involves the activation of phospholipases Cβ or γ (PLCβ

47 or PLCγ) (3). These proteins synthesize inositol-triphosphate (IP3) and diaglycerol (DAG) which

48 allows the activation of several protein kinase C (PKC) isoforms and their recruitment close to

49 PKD1. Once nearby, PKCs phosphorylate PKD1 onto two serine residues (738 and 742, or 744

50 and 748, human or murine numbering, respectively) localized in its activation loop leading to the

51 stimulation of the catalytic domain and its autophosphorylation onto its serine 910 (or 916 for

52 murine PKD1) residue (4). Activated PKD1 thus translocates into different cellular

53 compartments modulating its targets. The wide diversity of its substrates makes PKD1 a main

54 actor in several biological processes such as cell proliferation, migration, invasion, apoptosis,

55 angiogenesis, cardiac contraction, and immune regulation (5). In this context, its dysregulation

56 (over- or under-expression) was shown to be associated to diverse pathologies such as

57 inflammation, cardiac hypertrophy, and cancer (6). However, it remains largely unknown what

58 regulates PKD1 gene (prkd1) expression in tumors. PKD1 gene promoter was shown to be either

59 activated by the oncogenic KRas-NF-κB pathway increasing the expression of PKD1 in

60 pancreatic cancer cells (7) or inhibited by beta-catenin in prostate cancer (8). It was also shown

61 to be the target of epigenetic methylation decreasing PKD1 expression in some breast tumor cells

62 (9-11). These different molecular mechanisms lead to tumor tissue specific PKD1 mRNA

63 expression profiles According to TCGA data, PKD1 mRNAs are mostly expressed in prostate

64 cancer and melanoma. (Figure 1). Also, the data relative to 11 studies [Breast Invasive

65 Carcinoma, Colorectal Adenocarcinoma, Head and Neck Squamous Cell Carcinoma, Kidney

66 Renal Clear Cell Carcinoma, Kidney Renal Papillary Cell Carcinoma, Lung Adenocarcinoma,

67 Lung Squamous Cell Carcinoma, Pancreatic Adenocarcinoma, Prostate Adenocarcinoma, Skin

68 Cutaneous Melanoma, Stomach Adenocarcinoma (TCGA, PanCancer Atlas)], has shown PKD1

69 mRNA levels to be upregulated by 1.8 % (pancreas) to 18 % (lung adenocarcinoma) with a mean

70 value of 7.8 % (Additional files 1 to 8: Figures S1 to S8). Moreover, analysis of the prkd1 gene

71 reveals that only 4 % of the tumors analysed (206 patients over 5615) carry a mutation or a copy

72 number alteration in the 11 mentioned above TCGA studies (Figure 2). Taken together, these

73 results suggest, at least for tumors with the lowest upregulated values, that a dysregulated PKD1

74 activity may certainly play a more significant role in tumor progression than its gene

75 overexpression or amplification.

76 Although PKD1 seems to play an essential role in oncogenesis and is activated by a large variety

77 of stimuli, especially by many growth factors, it has a complex relationship with cell

78 proliferation both in normal and cancer cells. In fact, the specificity of the role of PKD1 with

79 regards to cell proliferation depends not only on the tissue type but also on the phenotype

80 (normal vs tumor) since PKD1 has been described to be either pro-proliferative or anti-

81 proliferative (Table 1). Moreover, this complexity drastically increases when, for a given cell

82 type, some controversial data exist. However, increasing data described PKD1 activity as

83 affecting tumor behaviour both in vitro and in vivo through its ability to regulate cell

84 proliferation making PKD1 a putative pertinent pharmacological target in oncology. In this

85 context, it becomes obviously and urgently crucial to have a clear knowledge of its role in cell

86 proliferation. Therefore, this review aims to list the major data existing to date concerning the

87 pro- or anti-proliferative effects of PKD1 and tries to bring elements of discussion to explain,

88 when necessary, potentially contradictory results.

90 PKD1 stimulates angiogenesis, a key determinant in cancer development.

91 Angiogenesis is the process by which new blood vessels are formed and is of pivotal importance

92 in processes such as wound healing and embryonic vascular development (12). It also plays a

93 fundamental role in tumor growth and metastasis (13). It provides tumors with oxygen and

94 nutrients, crucial for their developments and also helps in discarding tumor metabolites (13).

95 Inhibition of angiogenesis has thus been regarded as a valuable new approach to cancer therapy

96 (14). A number of stimulators and inhibitors regulate angiogenesis (15). In the case of PKD1, a

97 consensus exists with regard to its pro-angiogenic and thus pro-proliferative role whatever the

98 cell model (endothelial progenitor cells (EPCs) or cell lines) or the species (human or even

99 zebrafish). VEGF, a major component of angiogenesis under both physiological and pathological

100 conditions, induces the phosphorylation of PKD1 in human umbilical vein endothelial cells

101 (HUVEC) and bovine aortic endothelial cells (BAEC) (16) and EPCs (17). This occurs within

102 minutes upon binding of VEGF to its receptor, VEGFR2, through a PLCγ/PKCα-dependent

103 signaling pathway (16). VEGF-stimulated ERK1/2 phosphorylation and DNA synthesis in

104 HUVEC (16), and VEGF-induced microvessels sprouting from mouse aortic rings (18) were

105 markedly inhibited by PKD1 knockdown and PKD1 kinase negative mutant expression,

106 respectively, making PKD1 a pro-proliferative protein in endothelial cells. As previously

107 mentioned, histone deacetylases (HDAC) help control gene expression by regulating the

108 acetylation state of the chromatin. VEGF stimulates HDAC7 and HDAC5 phosphorylation and

109 their nucleocytoplasmic shuttling through a PKD1-dependent signaling pathway (17-19). Once

110 phosphorylated and in the cytosolic compartment, HDAC7 is localized away from its substrates

111 promoting gene expression leading to cell proliferation. Moreover, PKD1 was shown to regulate

112 the VEGF-induced expression of metalloproteinases such as MT1-MMP [13] which gene

113 expression is implicated in angiogenesis in vivo (20). Thus, it is very interesting to note that

114 PKD1 has a particular role with regard to VEGF since, on one hand, PKD1 is an actor of the

115 VEGF signaling pathway and, on the other hand, PKD1 regulates VEGF secretion as shown in

116 pancreatic cancer cells (21). Thus, in a tumor context, PKD1 could allow the activation of a self-

117 sustained loop allowing the formation of new vessels promoting the progression of the tumor

118 (22).

119

120 PKD1 promotes fibroblasts proliferation

121 The tumor microenvironment (TME) of cancer cells has been found to be a key determinant in

122 tumor progression and metastasis and has thus been gaining increasing interest in cancer

123 research. Fibroblasts, a major component of the TME, are responsible for the synthesis,

124 deposition and remodeling of the extracellular matrix and are a source of paracrine growth

125 factors that regulate the growth of cancer cells (23). PKD1 is portrayed as a pro-proliferative

126 protein in fibroblasts. Overexpression of PKD1 in murine Swiss-3T3 cells enhances the

127 proliferative response to G-protein coupled receptor agonists and to phorbol esters. In fact,

128 treatment with angiotensin, bombesin or phorbol 12,13-dibutyrate (PDBu) led to PKD1

129 phosphorylation onto its 744, 748 and 916 serine residues (murine numbering). Furthermore,

130 PKD1 overexpression potentiated neuropeptide-induced mitogenesis (24) probably through an

131 increased duration of the ERK signaling pathway characterized by a significant increase in the

132 phosphorylation of FAK and RSK, and by the accumulation of the early gene c-Fos (25).

133

134 PKD1 as a pro-proliferative and pro-survival factor in skin carcinogenesis and a putative

135 target in melanoma

136 Skin cancer is characterized by an abnormal growth of skin cells. Depending of the skin cell type

137 involved, two major categories of cancers were defined: basal and squamous cell skin cancer,

138 and melanoma (26). Basal and squamous cell cancers are the most common types and are mainly

139 caused by UV exposure, thus usually developing on body parts exposed to sunlight (27). On the

140 other hand, melanoma develops from melanocytes and has less well-defined origins. It can also

141 be caused by UV light but, unlike basal and squamous cell cancers, can also develop on body

142 parts unexposed to sunlight (28). Melanoma is more likely to form metastasis in other tissues

143 making it usually more aggressive than basal and squamous cell carcinomas (29).

144 PKD1 expression was first described in 1999 in mouse epidermis and positively correlated with

145 cell proliferation (30). Keratinocytes proliferation was decreased after treatment with PKD1

146 pharmacological inhibitor, Goedecke 6976 (Gö6976), and enhanced in PKD1-overexpressing

147 cells (30). Moreover, carcinoma (but not papilloma) from a two-stage carcinogenesis induced

148 mouse model, using first 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and then

149 tetradecanoylphorbol-13-acetate (TPA) as promoter, expressed high levels of PKD1 (30) and

150 were strongly impaired for their development after peracetylated EGCG (AcEGCG)-induced

151 PKD1 inhibition (31). According to these data, murine PKD1 was shown to be mostly expressed

152 in the basal, proliferative, layer of the epidermis and, although present, less expressed in

153 suprabasal layers (32). Consistently, overexpressing PKD1 in primary keratinocytes stimulates

154 keratin 5 (pro-proliferative marker) but inhibits involucrin (pro-differentiative marker) promoter

155 activities, respectively (32). Moreover, genetic PKD1 depletion not only inhibited cell

156 proliferation but also strongly potentiated the calcium-induced expression of late, intermediate,

157 and early differentiation markers of mouse keratinocytes such as loricrin, involucrin and keratin

158 10 (33). Finally, further evidence of a pro-proliferative role of PKD1 came from adenovirus-

159 transfected primary mouse epidermal keratinocytes showing that a constitutively active PKD1

160 mutant significantly increased DNA synthesis. By contrast, a dominant negative PKD1 mutant

161 inhibited it (34). Altogether, these results clearly established PKD1 as a pro-proliferative and

162 anti-differentiating protein in mouse keratinocytes. Nevertheless, one study reveals that these

163 pro-proliferative and anti-differentiative PKD1 functions would only be revealed in particular

164 situations. In fact, mice carrying conditional and specific disruption of PKD1 in keratinocytes

165 (K14-Cre-PKD1-cKO) displayed no alteration in epidermal proliferation and differentiation

166 suggesting that PKD1 would be dispensable for skin development and homeostasis under normal

167 conditions (35). However, PKD1-cKO-deficient mice displayed strong impaired wound healing

168 and re-epithelialization and became mostly totally refractory to DMBA/TPA-induced tumor

169 formation (35). These results are of crucial importance since they underline the role of PKD1 in

170 adaptive responses such as skin carcinogenesis and are finally in total accordance with data

171 showing that PKD1 was activated by UVB and that its overexpression protected keratinocytes

172 from UVB-induced apoptosis (36). In fact, through the stimulation of pro-survival signaling

173 pathways, PKD1 could thus allow the proliferation of mutated cells leading to cancer formation.

174 When considering normal human skin cells, the status of PKD1 expression remains more

175 elusive. In fact, PKD1 was initially described to be expressed throughout the superbasal layers

176 with a predominant expression in the stratum basalis, in accordance with its pro-proliferative role

177 as previously described in mice (37). However, further results indicated that PKD1 was not

178 detected in human keratinocytes, these cells being more dependent on the two other isoforms of

179 the PKD family, PKD2 (pro-differentiative), and PKD3 (pro-proliferative), for their proliferation

180 (38). As hypothesized by the authors, such discrepancies could be the consequence of a certain

181 lack of specificity of the antibodies used by Ristich et al. (37) since sc-935 antibody has been

182 reported by others to cross-react with PKD2 in western blot analyses (39). However, even if we

183 rely on this hypothesis, remains to be understood why the expression of the pro-differentiative

184 PKD2 would be the most abundant in actively dividing cells. In hyperplastic human skin

185 disorders, such as melanoma (40), basal cell carcinoma, and psoriasis (37) PKD1 was found to

186 be up-regulated. Taken together, these data suggest that, despite the fact that PKD1 seems not to

187 be of primary importance in normal skin homeostasis (35), it has, whatever the species models,

188 an evident pro-proliferative role in the context of skin carcinogenesis, wound healing and other

189 skin hyper-proliferative diseases like psoriasis. Consequently, these data also suggest why

190 targeting PKD1, by relatively selective inhibitors, has to be considered as an option for the

191 treatment and prevention of epidermal tumorigenesis, and for other hyper-proliferative diseases

192 such as psoriasis. To this goal, we showed that inhibition of PKD1 in melanoma cells 1)

193 decreased their colony forming capacities probably through the regulation of the ERK, JNK, and

194 NFκB signaling pathways, and 2) induced the relocation of β-catenin from nucleus to plasma

195 membrane, and the subsequent expression decrease of some pro-proliferative target genes such

196 as cyclin D1 (40).

197

198 PKD1 positively affects pancreatic inflammation and adenocarcinoma proliferation

199 Although PKD1 was shown to have a main role in insulin secretion of pancreatic islets (41), our

200 review will only focus on the exocrine function of the organ since tumors arouse mainly from

201 these structures. Among pancreatic cancers, the most common is the pancreatic ductal

202 adenocarcinoma (PDAC) representing 90 % of all cancers and considered among the most lethal

203 cancers with a very low 5-year survival rate of about 3-5 %. It is characterized by an early

204 metastatic state associated with a rapidly succeeding chemo resistance (42).

205 The expression status of PKD1 is often incorrectly formulated due to sentences that, using

206 shortcuts, become inaccurate in some publications. Indeed, stating that PKD1 is not expressed in

207 the normal exocrine pancreatic cells is incorrect since PKD1 was detected in untreated rat

208 pancreatic acini and very rapidly phosphorylated (detectable effect after 30 s) and activated by

209 cholecystokinin (CCK) through a PKC-δ dependent signaling pathway (43). PKD1 plays a major

210 role in rat pancreatic acini modulating experimental pancreatitis. Pharmacological inhibitors of

211 PKD1 attenuated early pancreatitis events (44) but also significantly attenuated pancreatic injury

212 when used as post-treatment (45). These results are of crucial importance since, by promoting

213 pancreatitis associated necrosis (46), PKD1 would promote a pro-inflammatory state, especially

214 characterized by the secretion of IL-6 and MCP-1 proteins (45), which could give rise to

215 pancreatic lesions, known as risk factors for cancer development. This is consistent with data

216 showing that PKD1 is upregulated in mice pancreatic acinar cells that undergo acinar-to-ductal

217 metaplasia (ADM) (47). ADM occurs after inflammation or injury and is reversible unless in a

218 persistent pro-proliferative context where it can progress to neoplasia and cancer (48, 49).

219 During ADM, PKD1 was shown to act downstream of TGFα and K-Ras, and upstream of the

220 Notch pathway, to promote the formation of ductal structures (47).

221 On the other hand, PKD1 is very moderately expressed in normal mouse and human pancreatic

222 tissue contrary to PKD3 that represents the major, if not the single, isoform (50). However,

223 transformed acinar cells and human PDAC show a strong increase in PKD1 expression

224 compared to normal tissue (7, 51, 52). PKD1 shortened the doubling time of PKD1-transfected

225 Colo357 cells by 20 % probably through an enhanced expression and activity of hTERT (52) and

226 dose-dependently increased DNA synthesis and cell proliferation of inducible PKD1-expressing

227 Panc-1 cells (53). The latter result is of main importance since it makes a clear proportional link

228 between PKD1 expression levels and cell proliferation rates. By strengthening the duration of

229 ERK signaling and inhibiting G protein-coupled receptors (GPCR)-induced c-Jun

230 phosphorylation, increased PKD1 levels stimulate cell cycle progression allowing the

231 accumulation of immediate gene products such as c-Fos, whereas inhibition of c-Jun

232 phosphorylation leads to the attenuation of the JNK-signaling switching its pro apoptotic action

233 to a pro-proliferative one (53). Another hallmark of PDAC is a highly increased NF-κB

234 signaling, linked to an increased proliferation of tumor cells. Oncogenic K-Ras induces canonical

235 NF-κB signaling and upregulates PKD1 expression and activity (7). Moreover, overexpression of

236 PKD1 increased anchorage-independent growth of PDAC cells (21), whereas its

237 pharmacological inhibition by CRT006001 (51), or the expression of a PKD1 kinase-dead

238 mutant (53), or its molecular silencing decreased the number of colonies formed in a semi-solid

239 medium (21). The pro-oncogenic role of PKD1 was further demonstrated in vivo since orally

240 given PKD1 inhibitor CRT0066101 significantly reduced the volume of established tumors in

241 subcutaneous Panc-1 xenograft models, or inhibited the final tumor volume of orthotopic

242 implanted Panc-1 cells (51). These results therefore highlighted the role of PKD1 not only in the

243 genesis but also in the maintenance of pancreatic tumors. These effects could be the consequence

244 of the regulation of angiogenesis. In fact, PKD1 expression stimulates the secretion of pro-

245 angiogenic factors such as VEGF and CXCL-8 and enhances the association between pancreatic

246 cancer cells and endothelial cells on Matrigel whereas the PKD1 inhibitor CRT0066101 reduces

247 angiogenesis in orthotopic PDAC tumor explant in vivo (21).

248 Taken all together, these data define PKD1 as a clear and prominent pro-proliferative factor in

249 PDAC making this protein a putative target for the development of new therapeutic strategies

250 against pancreatic cancer.

251

252 Pro- or anti-proliferative role of PKD1 in the gastro-intestinal tract, depending on the

253 localization

254 Despite the scarcity of studies, it seems that PKD1 plays different roles depending on which part

255 of the tract is considered. Compared to normal tissues, PKD1 expression was shown to be

256 markedly downregulated in gastric (11, 48, 54) and colorectal human cancer cells (55, 56), with

257 a more pronounced decrease in higher grade tumors (54, 55) suggesting an anti-oncogenic role of

258 this protein in these tissues. In fact, overexpression of PKD1 in human gastric adenocarcinoma

259 cells (AGS cells) (54) or in human SW480 colorectal cancer cells (55) inhibited cell

260 proliferation, clonogenicity, and motility, and delayed tumor growth in a xenograft mouse model.

261 Such effect could be dependent on PKD1-induced nuclear exclusion of β-catenin and the

262 subsequent decrease of its transcriptional activity (55) towards several proto-oncogenic genes

263 like cyclin D1 or c-Myc (57). Although these results were both provided upon PKD1

264 overexpression, potentially generating false very low level affinity interactions, and in a cell

265 model, SW480 cells, chosen upon its particular PKD1 and β-catenin expression and localization

266 levels, they suggest that PKD1 may negatively regulate cell proliferation through a β-catenin-

267 dependent mechanism in normal colorectal cells. Therefore, loss of PKD1 expression during

268 steps of tumorigenesis would release this break, promoting cell proliferation. Thus, down-

269 regulation of PKD1 expression levels appears as a key determinant for gastric and colorectal

270 tumorigenesis process and could be the consequence of an epigenetic inactivation occurring on

271 the PKD1 promoter as demonstrated in gastric cancer cells (11). However, this mechanism does

272 not seem to be generalizable to other parts of the gastro- intestinal tract since authors mentioned

273 that DNA methyltransferase inhibitors were unsuccessful to re-express PKD1 in colorectal

274 cancer cells (56).

275 By contrast, PKD1 was shown to have a pro-proliferative role in intestinal cells both in vitro and

276 in vivo suggesting a potential pro-oncogenic role in the intestine. Selective knockdown of

277 endogenous PKD1 inhibited DNA synthesis and cell proliferation induced by angiotensin II and

278 vasopressin in IEC-18 rat intestinal cell line (58). Moreover, overexpression of PKD1 in small

279 intestine of transgenic mice increased the proliferation rate and the number of intestinal cells per

280 crypt in vivo (58). PKD1 overexpression would enhance the proliferation induced by GPCR

281 agonists-dependent signaling pathways through the phosphorylation and the subsequent nuclear

282 export of class IIa histone deacetylase (such as HDAC4, HDAC5, and HDAC7) (59). In fact,

283 HDAC regulate gene expression by interacting with and repressing various transcription factors

284 (60). More recently, PKD1 overexpression was also shown to promote angiotensin II stimulated

285 cell proliferation by inducing β-catenin translocation to the nucleus (61). These intriguing results

286 were in total contradiction with previous ones found in human colon cancer cells (55),

287 highlighting the apparent complexity of the molecular mechanisms regulated by PKD1.

288 However, it is essential to notice that these contradictory results were 1) not conducted in the

289 same species (murine vs human) suggesting that PKD1 “species specific roles” cannot be

290 excluded; 2) performed either in tumor models, for colon and stomach, or in normal cells, for the

291 intestine, each expressing specific cellular contexts that could be determinant to define the role

292 of PKD1. Among the suspected proteins, the two other members of the PKD family are of

293 interest and most particularly PKD2. Indeed, PKD2 is often described to display opposite

294 functions to PKD1 and their respective expression is often inversely regulated as demonstrated in

295 colon (56) and gastric (54) cancers. Therefore, one may suppose that depending on the relative

296 expression level of PKD2, the apparent role of PKD1 could be consequently modulated.

297 Therefore, whenever possible, this point should be taken into consideration and further studies

298 are still needed to a better understand of the role of PKD1 in the whole gastro-intestinal tract.

299

300 PKD1 stimulates renal duct cells proliferation through a sustained ERK1/2 activation

301 Kidney cancer is among the tumors with the fastest growth rate and is the deadliest type of

302 urinary tract cancer (62). Aldosterone is a mineralocorticoid hormone that regulates ion fluxes

303 among nephron epithelium. In addition to its well characterized role as an ion transport

304 modulator (63), aldosterone was also shown to stimulate the proliferation of human RCC (renal

305 cell carcinoma) cell lines (64) as well as the murine M1 cortical collecting duct cell line (M1-

306 CCD) (65). In M1-CCD cells, aldosterone was shown to stimulate the phosphorylation of PKD1

307 through a mineralocorticoid receptor- (MR) and EGFR-dependent mechanism (66). PKD1

308 knockdown inhibited aldosterone stimulated proliferation demonstrating a pro-proliferative role

309 of PKD1 in this cell line (65). PKD1 may promote aldosterone-induced cell proliferation by

310 maintaining a sustained activation of ERK1/2 and inducing its translocation to the nucleus (65).

311 Taken together, these findings highlight the pro-proliferative role of PKD1 in renal collecting

312 duct cells. However, it is important to notice that among 17 tissues analyzed from TCGA data,

313 high PKD1 mRNA expression is a good prognostic factor in kidney tumors (Table 2). Despite

314 their apparent contradiction, these results mainly highlight, as previously mentioned in the

315 introduction, that a direct correlation between tumor mRNA expression levels and PKD1 activity

316 cannot assumed and that the relevant and interesting prkd1 gene expression analysis cannot be

317 freed from that of PKD1 activity.

318

319 PKD1 and the lung, a pro- or anti-proliferative role yet to be determined

320 Lung cancers are the leading cause of cancer mortality worldwide (67). Among the two subtypes,

321 non-small cell lung cancers (NSCLC) are the most common (85 %), whereas small cell lung

322 cancers (SCLC) are usually more likely to spread and become life threatening (68). Due to few

323 studies about PKD1 in the lung, it remains difficult to have a clear idea about the pro- or anti-

324 proliferative role of this protein in this tissue.

325 PKD1 was shown to be highly expressed and phosphorylated in bronchiolar and regenerative

326 alveolar epithelia from idiopathic pulmonary fibrotic patients (69). This pathology is

327 characterized by lung fibroblast activation and proliferation suggesting a pro-proliferative role of

328 PKD1 in this tissue (70). By contrast, PKD1 mRNA expression was shown to be down-regulated

329 in NSCLC compared to normal tissues especially when NSCLC patients displayed venous

330 invasion or lymph node metastasis, suggesting that PKD1 would negatively regulate NSCLC

331 tumor development (71). Consistent with this, although PKD1 was shown to induce a prolonged

332 activation of the ERK1/2 signaling pathway in Swiss-3T3 cells (see Chapter Fibroblast), PKD1

333 mediates the inhibition of PMA-induced ERK phosphorylation in A549 cells. Thus,

334 pharmacological PKD1 inhibition or its down-regulation resulted in enhanced PMA-induced

335 S6K1 and ERK phosphorylation and A549 cell proliferation whereas constitutively active PKD1

336 results in S6K1 and ERK inhibition (71). Interestingly, the anti-proliferative role of PKD1 may

337 also be dependent upon its ability to maintain a low-proliferative epithelial phenotype of lung

338 cells. In fact, PKD1 was also described to directly bind to E-cadherin leading to its membrane

339 redistribution and activation independently of DAG or PKC in A549 cells (72). Since PKD1

340 positively regulates E-cadherin transcription, this interaction/activation generates a positive

341 feedback loop favoring the maintenance of a low-proliferative epithelial phenotype. Conversely,

342 and in accordance with these results, knocking-down of PKD1 induces the loss of expression of

343 E-cadherin promoting the epithelial to mesenchymal transition and the acquisition of migratory

344 capacities (72).

345 However, it is important to note that in the study by Ni et al. (71) PKD1 protein expression levels

346 were not determined in NSCLC tissue specimens and even if mRNA levels decreased, no one

347 can conclude that protein levels will automatically follow the same profile. However, if these

348 results are also later confirmed at the protein level, future studies would have to carefully

349 consider these data to study the role of PKD1 in lung. Indeed, the use of the adenocarcinoma-

350 derived human alveolar basal epithelial cell model, A549 cells, cannot then represent a good

351 model enough for NSCLC investigations insofar as this tumor cell line expresses large amounts

352 of PKD1 (73) unlike what has been found in human tissues.

353

354 Are the pro-proliferative and pro-survival roles of PKD1 hormone-dependent in prostate

355 cancer cells?

356 Prostate cancer is the second leading cause of cancer death and the most commonly diagnosed

357 cancer in males in the U.S. (74). Despite advances in the screening methods, effective treatments

358 of advanced androgen-independent tumors are still to be found.

359 Scores of studies analyzed the roles of PKD1 in prostate cancer cells. PKD1 was shown to be

360 down-regulated in metastatic androgen-independent prostate cancers compared to their

361 respective primary tumor (75, 76). Moreover, PKD1 is highly expressed in low proliferative and

362 low metastatic androgen-sensitive LNCaP cells and down-regulated in the castration-resistant

363 LNCaP-derivative cell line, C4-2 cells (androgen-hypersensitive), or in the highly metastatic

364 androgen-insensitive DU145 and PC3 cells (75-77). These results suggested an association

365 between the down-regulation of PKD1 and the progression and aggressiveness of prostate

366 cancer. Knockdown of PKD1 using shRNA enhanced cell growth (77) whereas its

367 overexpression inhibited cell proliferation (77, 78). Moreover, curcumin was also thought to

368 inhibit prostate cancer cell proliferation through a PKD1-dependent mechanism (79). All these

369 results indicated that PKD1 can be considered as an anti-proliferative protein in prostate cancer

370 cells. Such an effect could be mediated through the binding of PKD1 with β-catenin and the

371 subsequent inhibition of β-catenin-mediated proliferation function (78), or through the secretion

372 of matrix metalloproteinase-2 and -9 (75). However, despite many studies, the precise function

373 of PKD1 with regards to cell proliferation and the mechanisms it controlled remain somewhat

374 unclear. For instance, inhibition of PKD1 expression drastically decreased ERK phosphorylation

375 in DU145 cells (75) although this protein is described as mediating pro-proliferative signaling

376 pathways (80). Moreover, the molecular mechanisms by which PKD1 would inhibit cell

377 proliferation have been mostly demonstrated in PKD1 overexpressing prostate cancer cells (75,

378 77, 78, 81). This technical approach is not necessarily the better way to proceed since a recent

379 study showed that according to TCGA data [Prostate Adenocarcinoma (TCGA, PanCancer

380 Atlas)], mRNA PKD1 expression levels in prostate cancer is upregulated in about 5 % tumors

381 suggesting that PKD1 hyperactivity may play a more important role in tumor progression than

382 overexpression (82). Moreover, LNCaP cells already express very high amounts of PKD1

383 making the relevance of such a model questionable insofar as overexpression can only lead to

384 nonspecific and non-relevant interactions. Furthermore, the comparison of results obtained in

385 different cell lines is also complicated and very hazardous. Indeed, the most commonly used cell

386 lines (i.e. LNCaP, C4-2, and PC3) display different sensitivities to androgens. However, PKD1

387 has a particular relationship towards androgen receptor, AR. Indeed, PKD1 would inhibit AR-

388 mediated transcriptional activity (observed in PKD1 and AR overexpressing cells) (81) while

389 androgens would inhibit PKD1 expression through the expression of a repressor, FRS2 (83).

390 Consistent with the latter results, incubation of cells in an androgen-depleted medium increased

391 PKD1 expression (83) indicating that AR expression and androgen sensitivity status of the cell

392 lines must be considered in a serious way and that the extrapolation of results between different

393 cell models cannot be done as easily as this. Therefore, even if these results should not be

394 questioned, one must be aware of their limits. In fact, it is interesting to note that contradictory

395 results exist even in studies conducted by the same team in which inhibition of PKD1 expression

396 was shown to have either an effect (77) or not (78) on the growth of LNCaP cells.

397 Although PKD1 was described as an anti-proliferative protein in prostate cancer cells, many

398 PKD1 targeting pharmacological inhibitory compounds were assayed in these cell lines (84-88).

399 Despite the fact that these molecules are not totally specific towards PKD1, they all induced cell

400 growth arrest that can be reversed through infection with adenovirus carrying PKD1 gene (87,

401 88) suggesting a pro-proliferative role of PKD1 that appears discrepant with data presented in the

402 previous paragraph. However, due to the drastic effects induced by these molecules on cell

403 growth, PKD1 appears more likely as a pro-survival factor as also suggested in LNCaP where

404 PKD1 was demonstrated to protect cells from PMA-induced apoptosis by promoting ERK and

405 NF-κB activities (84). Although an anti-proliferative effect is compatible with a pro-survival

406 role, these results indicate that more data are needed to better understand the role of PKD1 in

407 prostate cancer cells taking into account each particular cellular environment.

408

409 PKD1 expression is a poor prognostic factor in breast tumors

410 Breast cancer is the most common cancer, and the second leading cause of cancer-related death

411 in women (89). Despite improvement in early detection and treatment of breast tumors, advanced

412 metastatic breast cancer remains life threatening. Accumulating evidence show a potential role of

413 PKD1 in breast tumor progression. However, the link between PKD1 expression levels, PKD1

414 activation/activity and tumor aggressiveness remains unclear. In fact, PKD1 expression was

415 shown to be high in normal breast tissues and reduced in more than 95 % of invasive breast

416 cancer tissue and triple negative tumors (9, 90). Conversely, its expression is markedly increased

417 in breast cancer cell lines compared to normal cells where it is undetectable (91). Consistently, a

418 large-scale analysis performed in 152 malignant breast tissues showed that patients with poor

419 prognosis overexpressed PKD1, while those with good prognosis had significantly lower PKD1

420 expression levels (91) suggesting that PKD1 expression was positively linked to disease

421 progression. Although PKD1 expression was shown to be regulated through epigenetic

422 modifications such as DNA methylation of its promoter sequence (9), its expression status

423 should be more carefully studied in tissues and cell lines taking also into account the expression

424 levels of other determinant markers of breast cancers such as ERα, Her2 and progesterone

425 receptor (PR) since these proteins determine the upset of breast cancer progression.

426 Overall, PKD1 was shown to positively regulate cell proliferation. In fact, PKD1 overexpression

427 strongly and specifically increased MCF-7 cell growth by promoting G0/G1 to S phase transition

428 of the cell cycle through a MEK/ERK-dependent signaling pathway (92). Moreover, PKD1

429 overexpression improved anchorage- and growth factor-independent proliferation in vitro and

430 promoted tumor growth in vivo (92). It also increased ERα expression further demonstrating the

431 link that exists between these two proteins (91). Furthermore, PKD1 overexpression increased

432 MCF-7 cells sensitivity to estradiol, their independence towards estrogen for proliferation, and

433 their partial resistance to the antiestrogen, ICI 182,780 (91). Interestingly, this new cell behavior

434 looks like the one of prostatic C4-2 cells which, contrary to their parental cells, LNCaP, display

435 an androgen-independent and -hypersensitive phenotype for proliferation associated, in this case,

436 with a loss of PKD1 expression (see Chapter Prostate). More recently, our data were further

437 confirmed in the drug-resistant model of MCF-7-ADR cells expressing high levels of PKD1 as

438 well as cancer stemness markers compared to parental MCF-7 cells (93). In fact, knockdown of

439 PKD1 by siRNA- or microRNA-targeting PKD1 in MCF-7-ADR cells was shown to reduce the

440 number of tumor-spheres, to increase doxorubicin-induced apoptosis in vitro, as well as to

441 suppress tumor formation in xenograft models in vivo (93). Altogether, these results strongly

442 define PKD1 as a pro-proliferative and pro-tumorigenic factor in breast cancer cells.

443

444 PKD1 ectopic expression stimulates pre-T cell proliferation and is a potential molecular

445 target in EBV-associated B cell lymphoma

446 As proactive members of the tumor microenvironment, immune cells are main actors in tumor

447 progression since they can either positively or negatively regulate tumor growth depending on

448 their nature, activity and reciprocal interactions. In the perspective of developing anti-tumor

449 strategies, it is therefore relevant to know whether PKD1 regulates immune cell proliferation in

450 order to anticipate, as much as possible, the potential consequences of targeting PKD1 in tumors.

451 Studies have shown that PKD1 is not expressed in murine T- and B-lymphocytes, nor in

452 malignant B cells, nor in thymus and spleen (94-96), PKD2 being the major PKD isoform

453 expressed. However, ectopic expression of a constitutively active form of PKD1 induced pre-T

454 cell proliferation (97) illustrating again the pro-proliferative role of PKD1 when expressed and

455 raising questions about its putative function in hematopoietic malignancies. Nevertheless, PKD1

456 was shown not to be expressed in non-Hodgkin’ and Hodkin’s lymphoma (98) and, although it

457 regulates migration, PKD1 has no pro-proliferative role in multiple myeloma (99). Nonetheless,

458 Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) induces PKD1 expression in B cell

459 lymphoma and protects them from apoptosis. This contributes to the LMP-1-induced drug

460 resistance and progression of the pathology and makes PKD1 a potential molecular target in

461 EBV-associated B cell lymphoma (100).

462 Furthermore, cancer development is associated with a local inflammatory response that generally

463 surrounds the tumor. PKD1 has a dual role considering inflammation since it was shown either to

464 promote or inhibit inflammation through, among others, the secretion of chemokines by mast

465 cells (101-105). However, the use of distinct inflammation-inducing agents and methods to

466 analyze the inflammatory response makes it impossible to conclude, until now, about the role of

467 PKD1 in this physiopathological response.

468

469 Attempts to develop pharmacological agents altering PKD1 activity

470 Since PKD1 was mainly described as a pro-proliferative protein in several cancer cells both in

471 vitro and in vivo, it emerged as an interesting putative therapeutic target to fight against tumors

472 and different strategies were developed in the attempt to inhibit its activity. Among them,

473 different pharmacological inhibitors were developed and characterized as to their effectiveness

474 and specificity towards PKD1 both in vitro and in vivo (Table 3). Their inhibitory characteristics

475 (i.e. IC50) vary from one study to another since the experimental approaches used by authors to

476 determine them were not normalized. Three main techniques were commonly used such as the

477 measurement of the phosphorylation of a PKD1 substrate, mainly syntide 2 in an in vitro kinase

478 assay, the quantification of the cellular inhibition of the autophosphorylation of the PKD1 serine

479 910 (human numbering) residue analyzed by western immunoblotting (cellular inhibition of

480 PKD1 autophosphorylation) and the analysis of their effect on cell viability. Despite very

481 variable characteristics, these compounds were found to be effective in blocking proliferation

482 and other cellular functions such as invasion and migration of different cell models making them

483 promising inhibitors for cancer treatment (85). Unfortunately, they were also described to be too

484 rapidly metabolized, which limited their efficacy in vivo. However, among them, CRT0066101

485 was shown to inhibit growth of pancreatic, colorectal, bladder and triple negative breast cancer

486 cells xenografts in vivo (review in (106)) (107, 108). It thus appeared as a relatively good

487 therapeutic candidate since it blocked cell cycle progression at the G1 phase and increased

488 apoptosis by inhibiting the phosphorylation of “classical” pro-proliferative proteins such as Myc,

489 MAPK1/3, Akt, Yap, and Cdc2 (107). Studies have also suggested that the overexpression and

490 activation of PKD1 observed in CD34+ skin stem cells and skin tumors are potential targets for

491 the treatment of skin carcinogenesis (31). However, to our knowledge, no PKD1 inhibitors have

492 been used in clinical trials and further studies are absolutely necessary to notably increase the

493 specificity of such compounds toward not only the two other members of the PKD family, PKD2

494 and PKD3, but also toward other protein kinases in order to only interfere with PKD1-regulated

495 (or -dysregulated) signaling pathways. In fact, many of these compounds cannot be considered as

496 specific inhibitors of PKD1 and should be used with great caution in experiments concluding

497 about the specific role of PKD1 in several cell functions. To this end, molecular extinction of

498 PKD1 protein expression, when possible, remains a reliable technical approach to strengthen and

499 comfort results obtained with pharmacological compounds.

500

501 Concluding remarks

502 Whatever the cell type, the tissue, and its normal vs cancer status considered, it remains clear that

503 PKD1 plays a crucial role in growth-dependent signaling pathways. However, due to its potential

504 opposite functions, pro- or anti-proliferative, illustrated in Figures 3 and 4, respectively, the

505 development and the putative use of PKD1 targeting inhibitors as therapeutic tools may be

506 considered with major caution. Some contradictory data exist but are sometimes the

507 consequences of studies in which the analysis of the PKD1 phosphorylation level was too rapidly

508 correlated to the activity of the protein. But many results clearly indicated that such a

509 transposition cannot be made directly. Indeed, although the phosphorylation of PKD1 onto its

510 serine S738/742 and S910 residues seems to be important for its activation process, a direct

511 relationship between PKD1 activation and its catalytic activity is not always observed.

512 Consistently, the development of robust techniques allowing the direct and precise measurement

513 of PKD1 activity would be a major breakthrough in the field of PKD1 studies. Moreover, a very

514 recent study showed that a direct correlation can’t be automatically made between the relative

515 PKD1 expression levels between normal and tumor tissues and its role in tumorigenesis (109). In

516 fact, PKD1 is significantly down-regulated in head and neck localized tumors and metastases

517 compared to normal tissues due to epigenetic modifications, suggesting an anti-proliferative role

518 of this protein. However, its expression has been positively correlated with both the

519 subcutaneous head and neck squamous cell carcinoma xenografts growth and a sustained

520 bombesin-induced ERK1/2 activation demonstrating a pro-proliferative role of this protein (109).

521 In addition, despite numerous studies concerning PKD1, most of the data come from

522 experimental studies and very few information come from cohort ones. Thus, obtaining in vivo

523 data on large scales is also an important point to be considered in order to better understand the

524 role of this protein in the different tissues.

525

526 Grant Support

527 This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the

528 Ecole Normale Supérieure Paris-Saclay (ENS Paris-Saclay).

529

530 Acknowledgments

531 The authors thank Dr Sylvie Babajko for critical reading of the manuscript.

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756 positive breast cancer cell growth response to 17beta-estradiol and contributes to poor

757 prognosis in patients. J Cell Mol Med 2014;18:2536-52.

758 92. Karam M, Legay C, Auclair C, Ricort JM. Protein kinase D1 stimulates proliferation and enhances

759 tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling

760 pathway. Exp Cell Res 2012;318:558-69.

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768 lymphocytic leukaemia. Oncotarget 2016;7:41031-46.

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771 Commun 2016;7:12756.

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773 protein kinase D. Immunity 2003;19:491-501.

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775 Immunohistochemical studies of protein kinase D (PKD) 2 expression in malignant human

776 lymphomas. Exp Oncol 2006;28:225-30.

777 99. Qiang YW, Yao L, Tosato G, Rudikoff S. Insulin-like growth factor I induces migration and invasion

778 of human multiple myeloma cells. Blood 2004;103:301-8.

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780 malignant B cells from cell death induced by rituximab. Leuk Lymphoma 2015;56:194-201.

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782 by p38delta-PKD1 signaling in neutrophils confers inflammatory responses in the lung. J Exp

783 Med 2012;209:2229-46.

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785 proinflammatory response induced by hypersensitivity pneumonitis-causing thermophilic

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789 2017;198:4448-57.

790 104. Nielsen DSG, Fredborg M, Andersen V, Purup S. Administration of Protein Kinase D1 Induces a

791 Protective Effect on Lipopolysaccharide-Induced Intestinal Inflammation in a Co-Culture Model

792 of Intestinal Epithelial Caco-2 Cells and RAW264.7 Macrophage Cells. Int J Inflam

793 2017;2017:9273640.

794 105. Murphy TR, Legere HJ, III, Katz HR. Activation of protein kinase D1 in mast cells in response to

795 innate, adaptive, and growth factor signals. J Immunol 2007;179:7876-82.

796 106. Durand N, Borges S, Storz P. Functional and therapeutic significance of protein kinase D enzymes

797 in invasive breast cancer. Cell Mol Life Sci 2015;72:4369-82.

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799 Effective Drug for Treatment of Triple-Negative Breast Cancer. Cell Physiol Biochem

800 2019;52:382-96.

801 108. Li QQ, Hsu I, Sanford T, Railkar R, Balaji N, Sourbier C, et al. Protein kinase D inhibitor

802 CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell

803 cycle at G2/M. Cell Mol Life Sci 2018;75:939-63.

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825 Legends to Figures

826 Figure 1: Prkd1 RNA expression overview.

827 RNA-seq data in 9 cancer types are reported as median FPKM (number Fragments Per Kilobase

828 of exon per Million reads), generated by the The Cancer Genome Atlas (TCGA). RNA cancer

829 tissue category is calculated based on mRNA expression levels across all 9 cancer tissues and

830 include: cancer tissue enriched, cancer group enriched, cancer tissue enhanced, expressed in all,

831 mixed and not detected. Table presents the different representative values obtained for each

832 tumor.

833

834 Figure 2: Prkd1 gene mutation and copy number alterations.

835 Prkd1 genomic alteration types obtained by querying 5609 patients in 11 TCGA PanCan studies.

836 For each study, results are expressed as the frequency of alteration of the prkd1 gene compared

837 to the total number of cancers analyzed in the study.

838

839 Figure 3: Pro-proliferative signaling pathways regulated by PKD1.

840 Pro-proliferative signaling pathways modulated by PKD1. The schematic representation

841 illustrates how PKD1 may have pro-proliferative actions in various tissues. Activated PKD1 can

842 increase the duration of the ERK1/2 signaling pathway, leading in some tissues to a significant

843 increase in the phosphorylation of FAK and RSK, and the accumulation of the early-gene c-Fos.

844 PKD1 has also been described to regulate JNK and NF-κB activities. It can decrease the duration

845 of the JNK signaling thus diminishing c-Jun activity, and stimulate NF-κB signaling. PKD1 can

846 also induce the translocation of β-catenin from the plasma membrane into the nucleus where β-

847 catenin has a proliferative role through the induction of expression of pro-proliferative genes.

848 PKD1 was also shown to act upstream of and activate the Notch pathway. Moreover,

849 phosphorylation of HDACs (HDAC 4, 5 and 7) through a PKD1-dependent mechanism induces

850 their nucleocytoplasmic shuttling. Once in the cytosolic compartment, HDACs were away from

851 their target genes thus promoting gene expression and cell proliferation.

852 PKD1: β-cat: β-catenin, ERK: extracellular signal-regulated kinase, FAK: focal adhesion kinase,

853 HDAC: histone deacetylase, JNK: c-Jun N-terminal kinase, PKD1: protein kinase D1, RSK:

854 ribosomal S6 kinase.

855

856 Figure 4: Anti-proliferative signaling pathways regulated by PKD1.

857 Anti-proliferative signaling pathways modulated by PKD1. The schematic representation

858 illustrates how PKD1 may have anti-proliferative actions in various tissues. PKD1 can inhibit

859 downstream ERK and Akt pathways decreasing, in some tissues, S6K activity. PKD1 can inhibit

860 AR transcriptional activity. It can also induce the membrane redistribution of E-cadherin and β-

861 catenin, leading to the inhibition of the transcriptional activity of the latter. Furthermore, PKD1

862 can induce the expression of E-cadherin and metalloproteinases (MMP-2 and -9).

863 β-cat : β-catenin, ERK : extracellular signal-regulated kinase, MMP : matrix metalloproteinases,

864 PKD1 : protein kinase D1, S6K : ribosomal S6 kinase.

865

Table 1: role of PKD1 in normal and cancer cell proliferation

Tissue Cell type - cell line / Anti (-) or pro (+) Comments References Species proliferative effect Breast Breast cancer cell lines (human) - relative expression and invasion / metastasis (9) MCF-7 (human) + PKD1-overexpressing cells (91, 92) MDA-MB-415 (human) + (92) MCF-7-ADR (human) - drug resistant cells + (93) Endothelium HUVEC (human) + (16) cell migration (19) EPCs (human) + cell migration and tube formation (17) aortic ring (mouse) + microvessels sprouting (18) Zebrafish (in vivo) + (22) Fibroblast Swiss 3T3 (mouse) + (24, 25) NIH 3T3 (mouse) + PKD1-overexpressing cells (30) Head and neck HNSCC cell lines (human) and tissue sections + (109) squamous cells Kidney M1 (mouse) + (65, 66) Lung Idiopathic pulmonary fibrosis (human) + (69) A549, H520 - (71) Pancreas Acinar cells (rat) + NF-κB-dependent pancreatitis (45, 46) Rat (in vivo) + (46) Acinar cells (mouse) + acinar-to-ductal metaplasia, ADM (47) Human pancreatic adenocarcinoma + PKD1 expression (7, 52) Panc-1 and Panc-28 cells + WT and PKD1-overexpressing or -depleted cells (21, 51, 53) Colo357, PancTul, Panc89 + survival and telomerase activity (52) Prostate ALVA-41, LNCaP, C4-2, DU-145 - PKD1-overexpressing or -depleted cells (75, 77, 78, 81) LNCaP + pro-survival (84) LNCaP, PC3 + (85-88) PC3 + PKD1-overexpressing cells (88)

Tissue Cell type - cell line / Anti (-) or pro (+) Comments References Species proliferative effect Skin Primary keratinocytes (murine) + (30, 32) + PKD1-overexpressing or -depleted cells (33, 34, 36) Mouse (in vivo) + DMBA-induced tumors (31) + wound healing (35) Human basal cell carcinoma, psoriasis + (37) Melanoma (human) + (40) Stomach SNU gastric cell lines (human) - cell migration and invasion (11) AGS (human) - PKD1-overexpressing cells (54) Small Intestine IEC-18 (rat) + (58, 59, 61) PKD1 transgenic mouse (in vivo) + (58, 59, 61) Colon SW480 (human) - PKD1-overexpressing cells (55)

Table 2: PKD1 mRNA expression levels and survival analysis

Cancer Number of Alive Dead P value 5-year 5-year PKD1 as Correlation tumours analysed survival high survival low prognostic Melanoma 102 73 29 0.024 0%# 53%# No Glioma 153 30 123 0.21 10%# 8%# No Thyroid 501 485 16 0.00029 81% 95% Yes high expression is unfavorable Lung 1294 600 394 0.089 47% 44% No Liver 365 235 130 0.0011 26% 57% No Pancreas 176 84 92 0.071 33% 21% No Head and neck 499 281 218 0.19 43% 52% No Stomach 354 208 146 0.00095 22% 45% No Colorectal 597 473 124 0.023 55% 65% No Urothelial 406 227 179 0.39 38% 44% No Kidney 877 651 226 0.000024 75% 61% Yes high expression is favorable Prostate 494 484 10 0.16 98% 97% No Testis 134 130 4 0.20 100% 96% No Breast 1075 923 152 0.053 80% 88% No Cervical 291 220 71 0.32 64% 67% No Ovarian 373 143 230 0.053 28% 44% No Endometrial 541 450 91 0.067 73% 82% No

* Log-rank P value for Kaplan-Meier plot showing results from analysis of correlation between mRNA expression level and patient survival. # For melanoma and glioma, 3-year survival is shown.

Table 3: Inhibition of PKD1 by various compounds

Name IC50 (µM) Experimental approach to determine IC50 References 1-NA-PP1 0.155 ± 0.022 in vitro kinase assay (87) 22.5 ± 1.5 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (87) 23.3 ± 5.7 cell viability in PC3 cells (87) 1-NM-PP1 0.139 ± 0.033 in vitro kinase assay (87) 2,6-naphthyridines from 0.0004 to > 40 time-resolved fluorescence resonance transfer (TR-FRET) assay (110) 3,5-diarylazoles from 0.0037 to 6.9 time-resolved fluorescence resonance transfer (TR-FRET) assay (111) BPKDi 0.001 in vitro kinase assay (112) CID755673 from 15.510 ± 2.550 to 46.700 ± 27.650 colorectal cancer cell proliferation determined by (56) WST-1 assay (5 cell lines studied) 0.182 ± 0.027 in vitro kinase assay (85), (86), (113) 319.8 cell viability in PC3 cells (85) 0.5 ± 0.03 IMAP-based FP or TR-FRET kinase assays (86) 0.64 ± 0.03 IMAP-FP PKD1 (113) 11.8 ± 4.0 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (113) CID797718 7.0 ± 0.83 IMAP-based FP or TR-FRET kinase assays (86) 2.13 ± 0.21 in vitro kinase assay (86) 13.7 ± 0.42 IMAP-FP PKD1 (113) 2.34 ± 0.16 in vitro kinase assay (113) CRT0066101 from 0.770 ± 0.250 to 1.560 ± 0.340 colorectal cancer cell proliferation determined by (56) WST-1 assay (5 cell lines studied) 1.000 Panc-1 cell proliferation (BrdU) (51) 0.001 in vitro kinase assay (51) CRT5 0.001 in vitro kinase assay (114) 17 cell viability in HUVEC cells (114) Gö6976 0.020 in vitro kinase assay (115) IKK-16 0.154 ± 0.008 in vitro kinase assay (87)

Name IC50 (µM) Experimental approach to determine IC50 References K252a 0.007 in vitro kinase assay (115) kb-NB142-70 0.026 ± 0.006 in vitro kinase assay (116) 77.970 cell viability in endothelial cells (56) from 2.820 ± 0.67 to 8.35 ± 4.31 colorectal cancer cell proliferation determined by WST-1 assay (5 cell lines studied) (56) 2.2 ± 0.6 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (85), (113) 0.0283 ± 0.0023 in vitro kinase assay (85), (113) 8.025 cell viability in PC3 cells (85) 0.71 ± 0.02 IMAP-FP PKD1 (113) kb-NB165-09 0.0825 ± 0.005 in vitro kinase assay (85), (113) 3.1 ± 0.5 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (85), (113) 49.98 cell viability in PC3 cells (85) kb-NB165-31 0.114 ± 0.024 in vitro kinase assay (85), (113) 8.6 ± 2.0 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (85), (113) 31.91 cell viability in PC3 cells (85) kb-NB165-92 0.111 ± 0.006 in vitro kinase assay (85), (113) 2.6 ± 0.7 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (85), (113) 78.259 cell viability in PC3 cells (85) kb-NB184-02 0.193 ± 0.027 in vitro kinase assay (85), (113) 18.6 ± 2.0 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (85), (113) 33.84 cell viability in PC3 cells (85) SD-208 0.106 ± 0.006 in vitro kinase assay (88) 17.0 ± 1.5 cellular inhibition of PKD1 autophosphorylation (in LNCaP cells) (88) 17.0 ± 5.7 cell viability in PC3 cells (88) VCC251801 0.028 ± 0.002 in vitro kinase assay (116) 10.120 cell viability in endothelial cells (116)

Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. median FPKM (number Fragments Per Downloaded from Kilobase of exon per Million reads) Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. 10 15 20 25 0 5 Author ManuscriptPublishedOnlineFirstonJuly16,2019;DOI:10.1158/1541-7786.MCR-19-0125 mcr.aacrjournals.org 3 1 rd st median quartile quartile max min Figure 1: cancer on October 1,2021. ©2019 American Association forCancer Research. Lung 0.1 0.8 1.5 2.5 4.9

Stomach cancer Prkd1 0.4 0.7 1.3 2.6 0

Colorectal cancer 0.3 0.6 1.1 2.3 0

RNA expression overview

Melanoma 12.4 0.1 2.7 4.9 6.8

Headand cancer neck 0.2 0.4 0.9 0 2

carcinoma invasive Breast Breast 0.6 1.1 1.6 3.1 0

cancer Renal 0.2 2.6 3.9 5.5 9.9

Prostate cancer 22.6 1.2 6.5 9.5 13

Pancreatic cancer

0.1 1.5 2.3 3.2 5.7

Author Manuscript Published OnlineFirst on July 16, 2019; DOI: 10.1158/1541-7786.MCR-19-0125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

9 Multiple alterations

8 Deep deletion

7 Amplification

6 Fusion

5 Mutation

Alteration frequency (%) frequency Alteration 3

Figure 2 : Prkd1 gene mutation and copy number alterations Downloaded from mcr.aacrjournals.org on October 1,observed 2021. © 2019 American in 206 Association tumors for Cancer over Research. 5615 tumors analysed

Author Manuscript Published OnlineFirst on July 16, 2019; DOI: 10.1158/1541-7786.MCR-19-0125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

pancreas PKD1 endothelium skin Notch kidney prostate ERK1/2 β-cat JNK NF-κB breast +++ fibroblast intestine ERK1/2 P HDAC

β-cat FAK RSK ↓c- Jun

↑c-Fos ↑c-Fos NF-κB HDAC ↑c-Fos ² NUCLEUS

Figure 3: Pro-proliferative signaling pathways regulated by PKD1

MMP MMP lung MMP

stomach PKD1

prostate ERK

Akt ↓S6K E-cad E-cad PKD1 E-cad β-cat

β-cat AR ² NUCLEUS

Figure 4: Anti-proliferative signaling pathways regulated by PKD1

Deciphering the role of protein kinase D1 (PKD1) in cellular proliferation

Ilige Youssef and Jean-Marc Ricort

Mol Cancer Res Published OnlineFirst July 16, 2019.

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