Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet clinical need. Given the elevated ATP levels seen in PDAC, the purinergic axis represents an attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, it plays essential roles in fibrosis, inflammation response and immune function. We have analysed the PDAC purinome using publicly available databases to discern which members may impact patient survival. We identified P2RY2 to be the purinergic gene with the strongest association to hypoxia, the highest cancer cell specific expression and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification or pharmacological strategies we identify the mechanism of this ATP-driven invasion to require direct protein-protein interactions between P2Y2 and αV integrins. Using DNA-PAINT super-resolution fluorescence microscopy, we found that P2Y2 regulates the amount and distribution of integrin αV in the plasma membrane. This work highlights a novel GPCR-integrin interaction in cancer invasion and its potential for therapeutic targeting.


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Pancreatic ductal adenocarcinoma (PDAC), which accounts for 90% of diagnosed pancreatic 27 cancer cases, has the lowest survival rate of all common solid malignancies. Surgery is the 28 only potentially curative treatment, but unfortunately more than 80% of patients present with 29 unresectable tumours (Kocher, 2022). Consequently, most patients survive less than 6 30 months after diagnosis, resulting in a 5-year survival rate of less than 5% when accounting 31 for all disease stages (Bengtsson, Andersson and Ansari, 2020; Kocher, 2022). Despite 32 continued efforts, this statistic has improved minimally in the past 50 years. Due to increasing 33 incidence, late detection and lack of effective therapies, pancreatic cancer is predicted to be 34 the second most common cause of cancer-related deaths by 2040 (Rahib et al., 2021). 3 Extracellular ATP is known to promote inflammation (Kurashima et al., 2012), growth (Ko et 48 al., 2012) and cell movement (Martinez-Ramirez et al., 2016). Contrastingly, adenosine is 49 anti-inflammatory and promotes immunosuppression (Schneider et al., 2021). There are 50 ongoing clinical trials in several cancers, including PDAC, for drugs targeting the 51 ectonucleotidase CD73 (NCT03454451, NCT03454451) and adenosine receptor 2A 52 (NCT03454451) in combination with PD-1 checkpoint inhibitors and/or chemotherapy. 53 However, a Phase II multi-cancer study evaluating an anti-CD73 and anti-PD-L1 combination 54 was withdrawn due to minimal overall clinical activity (NCT04262388). This highlights the fact 55 that further mechanistic understanding of purinergic signalling in PDAC is required to exploit 56 its full therapeutic potential. 57 Here we combine bioinformatic, genetic and drug-based approaches to identify a novel 58 mechanism mediating ATP-driven invasion, uncovering a new therapeutic target in PDAC. 59 Beginning with an in-depth in silico analysis of the purinome of PDAC, using publicly available 60 patient and cell line databases, we build on bioinformatic data associating the purinergic 61 receptor P2Y2 with PDAC. After validating expression of P2Y2 in human PDAC cancers, we 62 focused on identifying the function of the receptor in cancer cells. In vitro data underlined the 63 importance of P2Y2 as a strong invasive driver, using a 3D physio-mimetic model of invasion. 64 Finally, using a super-resolution imaging technique, DNA-PAINT, we characterise the genetic alterations in 0-3% of tumours and a heterogeneous percentage of tumours with high 83 mRNA expression (z-score > 1) for each purinergic gene. 84 Since purinergic signalling has been strongly associated with hypoxia, the Winter (Winter et 85 al., 2007), Ragnum (Ragnum et al., 2015) and Buffa (Buffa et al., 2010) hypoxia scores were 86 used to examine the correlation between the expression of purinergic genes and HIF-1α in 87 the PAAD TCGA database (Sup. Fig. 1B). Samples were divided into low (n=88) or high 88 (n=89) hypoxia score, using the median hypoxia score to perform a differential expression 89 analysis. CD73 (NT5E), adenosine A2B receptor (ADORA2B) and P2Y2 (P2RY2) mRNA 90 expression associated strongly with the high hypoxia score group for all three hypoxia scores 91 (log2 ratio >0.5, FDR <0.001). P2Y2 had the highest log2 ratio in all hypoxia signatures 92 compared to other purinergic genes. With a more extensive gene signature, the Winter 93 hypoxia score (99 genes) allowed for a more comprehensive relative hypoxia ranking of 94 tumour samples, compared to Ragnum (32 genes) and Buffa (52 genes) signatures. Hence, 95 we used cBioPortal to generate a transcriptomic heatmap of purinergic genes, ranked using 96 5 the Winter hypoxia score and overlaid with overall survival data (Fig.1C). We observed a 97 direct correlation between Winter hypoxia score and decreased overall survival for high 98 hypoxia score-related purinergic genes. 99 We hypothesised that genes related to high hypoxia scores would be expressed preferentially 100 in the tumour core. Mining published RNA-seq data from 60 paired PDAC samples of stroma 101 and tumour microdissections (GSE93326) (Maurer et al., 2019) and performing differential 102 expression analysis, we observed that most genes related to high Winter hypoxia scores 103 (P2RY2, ADORA2B and NT5E) were expressed in the tumour epithelial tissue (Fig. 1D), 104 except for PANX1, encoding for pannexin 1, which is involved in cellular ATP release (Bao,105 Locovei and Dahl, 2004).

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To elucidate the cell type-specific purinergic expression landscape, we used published data biggest impact on adverse patient survival (Fig. 1B). These independent in silico analyses 120 encouraged us to explore the influence of P2Y2 on pancreatic cancer cell behaviour.

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To validate our bioinformatic findings, we performed RNAscope in human PDAC samples     153 To evaluate the impact of P2Y2 in pancreatic cancer cell invasion, we used a 3D hanging  Importantly, a non-hydrolysable ATP (ATPγS ;100 µM) showed similar effects, implicating 165 ATP and not its metabolites as the cause of the invasion (Sup. Fig. 3B). Of note, IF staining 166 8 of PS-1 cells showed negligible expression of P2Y2 (Sup. Fig. 3C). To determine whether 167 integrin association was necessary for ATP-driven invasion, we treated spheres with 10 µM 168 cyclic RGDfV peptide (cRGDfV), which binds predominantly to αVβ3 to block integrin binding 169 to RGD motifs (Kapp et al., 2017), such as that in P2Y2 (Ibuka et al., 2015). cRGDfV treatment 170 reduced ATP-driven motility significantly, both in 3D spheroid invasion assays (p < 0.001) 171 (Fig 3B, C) and in 2D Incucyte migration assays (Sup. Fig. 3D, E). Treatment with AR-C 172 decreased motility. To ensure that this behaviour was not restricted to AsPC-1 cells, 173 experiments were corroborated in the BxPC-3 cell line (Sup. Fig. 3F, G).

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To further verify that ATP-driven invasion was dependent on P2Y2, we silenced P2Y2 transfected with an empty vector (EV) or with P2Y2 RGE (Fig. 3I,J). Only when transfecting 191 PANC-1 cells with P2Y2 RGD was ATP-driven invasion observed (p < 0.001). These results 192 demonstrate that the RGD motif of P2Y2 is required for ATP-driven cancer cell invasion.  Moreover, many genes sets associated with P2Y2 expression were related to integrin 302 signalling. The RGD motif in the first extracellular loop of P2Y2 results in a direct interaction 303 of P2Y2 with RGD-binding integrins, particularly integrins αVβ3 and αVβ5 (Erb et al., 2001).

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This can exert phenotypic effects -for example, binding of P2Y2 to integrins via its RGD motif 305 is necessary for tubule formation in epithelial intestinal cell line 3D models (Ibuka et al., 2015). 306 We focus here on the importance of the RGD motif of P2Y2 in a cancer context. Treating 3D 307 PDAC spheroids with ATP resulted in increased invasion (Fig. 3), which was blocked by the  Despite efforts, there are currently no clinically efficacious P2Y2 antagonists, with poor oral 314 bioavailability and low selectivity being major issues (Neumann et al., 2022). Our findings 315 demonstrate that P2Y2 can also be targeted by blocking its interaction with RGD-binding 316 integrins, due to its dependence on integrins for its pro-invasive function. normal AsPC-1 cells with cRGDfV and ATP. We speculate that the RGD in P2Y2 may regulate 344 integrin-dependent P2Y2 internalisation and downstream signalling (Fig. 5).

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In summary, our study demonstrates that P2Y2, via its RGD motif, has a pivotal role in ATP-     considered. This histogram was fitted with a multi-peak Gaussian function to determine the 582 qPAINT index for a cluster of single molecule localisations corresponding to one protein (qi1).

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The calibration value obtained with this method was used to estimate the number of P2Y2