GNA15 signaling facilitates the initial phases of pancreas cell transformation and is associated with the basal-like/squamous subtype

doi:10.21203/rs.3.rs-5186783/v1

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GNA15 signaling facilitates the initial phases of pancreas cell transformation and is associated with the basal-like/squamous subtype

https://doi.org/10.21203/rs.3.rs-5186783/v1

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Curative intervention of pancreatic ductal adenocarcinoma (PDAC) remains substantially precluded because cancer cells typically spread asymptomatically before diagnosis.

We previously described GNA15 ectopic expression in neoplastic and pre-neoplastic PDAC lesions. Here, we show that GNA15deletion in a mouse model of Kras-dependent PDAC reduced pancreatic neoplastic lesions. Several studies stratified PDAC patients in the “classical/progenitor” and the “basal-like/squamous” molecular subtypes. We find GNA15 expression strongly associated with the “basal-like/squamous” subtype. Bioinformatic data and experimental results from PDAC cell lines and PDX revealed a gene signature implicated in cell-cell or cell-matrix interactions and invasiveness. GNA15 loss-of-function in PDAC cell lines promoted aggregation and reduced the expression of genes supporting cell invasion, such as PLAUR and FN1. Recently, the observation that cells belonging to both subtypes co-exist in the same patient was interpreted as the clonal evolution of the disease from the “classical/progenitor” to “basal-like/squamous”. The simultaneous association of GNA15 with early PDAC stages and the “basal-like/squamous” phenotype challenges this sequential progression while supporting a role for GNA15 in the early asymptomatic dissemination of the disease. The GNA15 signature could contribute a highly specific combination of bio markers and therapeutic targets to trace and eradicate the cellular component responsible for PDAC lethality.

Cell Communication and Signaling

GNA15

pancreatic adenocarcinoma

cell signaling

preneoplastic lesions

PDAC incidence continues to increase while effective treatments are still lacking. Only 15% of patients are diagnosed with localized disease and treated surgically but even in these cases undetected cells, that have already disseminated, cause relapse within two to five years ¹.

Gene expression profiles distinguished two major PDAC subtypes: “classical/progenitor” and “squamous”/basal-like/quasi-mesenchymal with implications for the choice of therapeutic approaches ^2–4. Disease etiology was interpreted to initiate with precancerous acinar-to-ductal metaplasia (ADM), progress to pancreatic intraepithelial neoplasia (PanINs) and/or intraductal papillary mucinous neoplasm (IPMN) and advance to PDAC with more aggressive cancer associated with squamous features and worse prognosis. Recent studies show that most tumors display a mixture of “basal-like/squamous”- and “classical/progenitor”-like cells ⁵. The origin or plasticity between the two subtypes ⁶ remains unclear, however hepatic metastases display higher “basal-like/squamous” profile than does the primary tumor ⁷.

Squamous features are common among other carcinoma originating in different organs and share common histopathological features, including the presence of keratins, tonofilament bundles, desmosomes and hemidesmosomes ⁸. These structures orchestrate cell-cell interactions with neighboring cells and are dynamically regulated during cell migration. This appears particularly relevant in PDAC because asymptomatic diffusion of the disease occurs well before diagnosis, preventing curative treatment ⁹. A better understanding of the molecular mechanisms that drive the asymptomatic diffusion of cells is key to identifying early and curative intervention.

With the exceptions of the esophageal mucosa and hematopoietic cell lineages, all healthy human tissues express virtually no GNA15. Ectopic GNA15 expression was observed in PDAC lesions and their precursors, PanIN and IPMN ¹⁰. The GNA15 gene encodes Gα15, a Gq class α subunit of heterotrimeric G proteins ^11,12. Gα15 can potentially transduce a broad array of G protein coupled receptor (GPCR) to signaling oncogenes relevant to PDAC ¹³ and we previously demonstrated it promotes viability ¹⁴, migration and invasiveness ¹⁵ of PDAC cell lines.

We hypothesize Gα15 signaling promotes PDAC oncogenesis. Here we report data from patients, cell lines and mice that converge to support a role for Gα15 signaling in the development of PDAC precursor lesions resetting extracellular interactions. The most intriguing implication is that the association to GNA15 anticipate the onset of the basal-like/squamous subtype in the initial and asymptomatic phases of PDAC progression.

2.1 Gene expression

PDAC and RNAseq derived data were validated on total RNA extracted from xenografted human PDAC lesions or PANC-1 cells respectively. RNA was isolated using TRIzol reagent (Life Technologies) following manufacturer’s instruction and quantified using Nanodrop (Thermo Fisher Scientific). 1 µg RNA was reverse-transcribed with High Capacity cDNA Reverse Transcription Kit (4368814) following manufacturer’s instruction and 1/10 of the reverse transcription was subjected to qPCR reactions. mRNA was quantified by TaqMan assay (Applied Biosystems Life Technologies, Carlsbad, CA ) performed in an ABI Prism 7900 HT instrument using the Universal PCR Master Mix (4304437) and assays for GNA15 (Hs00157720_m1), TACSTD2 (Hs05055338_s1), GJB3 (Hs02378125_st), CDH3 (Hs00999915_m1), CDCP1 (Hs1080405_m1), PEDS1 (Hs0419114_st), LY6E (Hs00158942-m1), ING3 (Hs00219444_m1), MDM2 (Hs01066930_m1), TP53 (Hs01034249_m1), SMAD4 (Hs0092647_m1), PLAUR (Hs00958880_m1), FN1 (Hs01549976_m1). All PCR reactions contained primers and probe diluted 1:20 and 5 ng cDNA (total RNA equivalent) in 25 µl total volume, and samples were analyzed in triplicate. Thermal cycling included an initial 95°C for 10 min then 40 cycles of 15 s at 95°C for denaturation and 1 min at 60°C for extension. As endogenous reference, B2M (Hs00187842_m1) and ACTB (Hs99999903_m1) transcript levels were assayed in parallel. The relative mRNA expression of each sample was calculated by the standard curve method according to Lai et al ¹⁶.

2.2 RNAseq differential expression analysis

RNA was isolated from TRIzol to prepare directional poly-A libraries diluted in NEBNext 500/550 High Output Reagent Cartridge V2 75 cycles (Illumina). Sequencing was performed on a Chip Bioanalyzer_6000 Nano QC RNA (Agilent). Differential expression analysis was performed using Partek software, reads were aligned using Bowtie, quantified, normalized and compared by DeSeq2 combining knockout clones to control samples.

2.3 Cell Culture

PT45 and PANC-1 cells were cultured in DMEM (10938025) supplemented with 10% fetal calf serum (FCS 10270106) and 1% pen-strep (15140122) all from Thermo Fisher Scientific. Cells were maintained at 37°C in a humidified incubator with 5% CO2. Cell culture medium was changed every 3–5 days depending on cell density. For routine passage, cells were split at a ratio of 1:5–10 when they reached 85–90% confluence. GNA15 knockout and knockdown were obtained by CRISPR/Cas9 and lentiviral particles respectively, as previously described ¹⁴.

2.4 Western Blot Analysis

Whole cell protein extracts were prepared in lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich), and centrifuged at 4°C for 10 min at 14,000 g. Protein concentration was measured by the Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). 30–40 µg of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline Tween-20 (TBST) for 1 h at room temperature and incubated with primary antibodies (pERK42/44 T183, Sigma-Aldrich M7802 (1:1000); p44/42 MAPK (Erk1/2), Cell Signaling 9102 (1:1000); Anti human Trop-2, R&D systems AF650 (0.2ug/mL); Anti-Vinculin 7F9, Sigma-Aldrich MAB3574 (1:1000); α-tubulin, Cell Signaling 2144 (1:1000)) in 5% nonfat dry milk in TBST at 4° C overnight followed by incubation with secondary antibody (Anti mouse IgG, ThermoFisher scientific 31430 (1:10000); Anti Rabbit IgG, ThermoFisher scientific 31460 (1:10000); Anti goat IgG, ThermoFisher scientific 81-1620 (1:10000)) in 5% nonfat dry milk in TBST for 1 h at room temperature.

ECL (Millipore, WBLUF0500) followed by exposure with Uvitec Alliance (Uvitec, Cambridge, UK) was used for protein band detection.

2.5 Flow Cytometry

Cells were collected by trypsinization and stained with PE Mouse Anti-Human Trop-2 antibody Clone 162 − 46 (BD Biosciences) for 20 min at 4°C. Analysis was conducted using FlowJo software.

2.6 Cell Aggregation assay

Cell-cell aggregation was analyzed as previously described ¹⁷. A preparation of a 6.7 mg/ml semi-solid agar medium was made by dissolving Bacto-agar (Agar Bacteriological OXOID) in sterile Ringer’s salt solution at 40–50 ̊C. 100 µL were transferred into each well of a 96-well microtiter plate and left at 4 ̊C to solidify for about 1 hour. Cells were detached by standard trypsinization procedures. A suspension of 40,000 cells, previously stained with a Hoechst’s 33342 solution (final concentration 1:500) for 20 minutes at 37 ̊C, was plated in each well. Cell aggregation was quantified by video time-lapse microscopy using EVOS-FL auto microscope (Life technologies) and FiJI software (NIH).

2.7 Mouse model

This study is performed in accordance with relevant guidelines and regulations and was approved by the Italian Ministry of Health authorization 308/2019-PR. All methods are reported in accordance with ARRIVE guidelines. Both male and female mice, KC (KrasG12D, Pdx1 Cre) and KCG (KC over a Gna15 knockout background) mice were maintained on a 12-hour day:12-hour night cycle and fed ad libitum with normal chow. Mice were sacrificed from 39–43 days of age, pancreata were collected with a piece of spleen and duodenum (with ampula of Vater) as landmarks for the head and tail of the pancreas and processed for paraffin embedding. 5 µm sections were stained with haematoxylin and eosin (H&E) by standard methods. Three to five sections were prepared for each mouse, separated by about 150µm. The sections were analyzed blinded by at least two of the authors by microscope using a 10x magnification object lens. Lesions were counted once each in nonoverlapping fields in a sweeping Z pattern from head to tail. Individual lesions were also visualized at higher magnification to confirm morphologic features.

2.8 Statistical analysis

The measurement values were presented as means ± SEM. Experiments involving western blot and RT-PCR techniques were repeated at least three times as defined in the figure legends. Statistical significance of differences in the measured mean frequencies between two or more experimental groups was calculated using the Student two-tailed t-test, Dunnett’s, Mann Whitney or ANOVA tests as described in the figure legends. Survival percentages were estimated using Kaplan-Meier methods, and survival curves were compared using the log-rank test.

2.9 Data and materials availability

The dataset for Pancreatic Cancer (“TCGA, Firehose Legacy”, “CPTAC, Cell 2021”) and Pan-cancer analysis of whole genomes (“ICGC/TCGA, Nature 2020”) of The Cancer Genome Atlas (TCGA, https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) were queried through the cBioPortal (¹⁸, https://www.cbioportal.org/), to study GNA15 mRNA expression in affected human tissues. PANC-1 RNAseq data are accessible in https://datadryad.org doi:10.5061/dryad.pg4f4qrw9.

3.1 GNA15 expression in cancer cells is associated with squamous features

Squamous cells are absent in normal pancreas but emerge when aberrant ductal-to-squamous transition alters cell fate and drives oncogenesis ¹⁹. GNA15 is ectopically induced in pancreatic neoplasia and is associated with more aggressive oncogenesis ¹⁰. A survey of human gene expression data for the multiple cancers of TCGA-ICGC shows that GNA15 expression is strictly associated to the five squamous cell carcinomas (Fig. 1A).

Within this subset of malignancies, GNA15 expression positively correlates with the expression of TP63 ²³ (Fig. 1B) a master regulator of the normal squamous lineage and the transformed basal-like/squamous subtype of PDAC ²⁴. Consistently, in TCGA-PAAD cohort, TP63 and GNA15 are overexpressed in the basal-like/squamous transcriptional subtype (Fig. 1C). The basal-like/squamous subtype has also been defined as “glycolytic”, in contrast to the “lipogenic” ‘classical/progenitor’ type ^3,25. We find GNA15 expression is proportional to hypoxia scores (Winter’s ²⁰, Ragnum ²¹ and Buffa ²²) based on markers like GAPDH (Fig. 1D) and others specific of the glycolytic component of the basal-like/squamous subtype (Supplementary Table 1). The hypoxia score was reported to be similar in parent lesions in the primary tumor versus matched daughter metastatic lesions and it correlated with a higher frequency in bi-allelic loss of the oncosuppressor TP53 independently of the microenvironment or a particular clinical or genetic feature ²⁶. Recently the loss of TP53 heterozygosity was hypothesized to characterize pre-tumor cells predisposed to progress towards subclonal evolution ²⁷. Thus, hypoxia-adaptation is likely to represent an inherent characteristic of the basal-like/squamous subtype, rather than a transient response to external conditions. Epigenetically driven GNA15 expression could be part of this early adaptation; it is not correlated with the presence of most prominent driver mutations in PDAC (Supplementary Fig. 1) but is directly correlated with loss of heterozygosity of TP53 and inversely correlated to the expression of the oncosuppressor SMAD4 (Fig. 1E, Supplementary Table 2) reported to be significantly reduced in the basal-like/squamous subtype ⁴.

To better understand the significance of GNA15 ectopic expression we analyzed TCGA-PDAC for co-expressed genes.

3.2 GNA15 expression is associated with an expression signature related to cell-cell interactions

Most genes co-expressed with GNA15 (Fig. 2A) have been previously reported to play a role in PDAC development (Supplementary Table 2). These associations were validated by RNAseq (Fig. 2B) and by RT-PCR (Fig. 2C) in 22 PDAC patient-derived xenografts (PDX).

Strengthening GNA15 expression as an indicator of poor prognosis ¹⁰, all positively correlated genes predicted poorer prognosis, opposite to negatively correlated genes (Supplementary Fig. 2). Gepia 2.0 analysis of neoplasia datasets present in TCGA reveals that GNA15 signature predicts earlier relapse in PDAC as compared to other cancers. The four genes most highly correlated to GNA15 expression encode transmembrane proteins. These genes comprise a PDAC specific restricted signature with a significant hazard ratio (Fig. 2D) that predict disease free survival comparable to a larger signature of 200 co-expressed genes with a Pearson score over 0.5, Fig. 2E and Supplementary Fig. 2B. These four genes could thus serve as prognostic biomarkers.

The gene most highly correlated with GNA15 expression is tumor-associated calcium signal transducer 2 (TACSTD2), the homolog of EpCAM that encodes the trophoblastic cell surface antigen 2 (Trop-2). Histological analysis confirmed the co-localization in neoplastic lesions (Supplementary Fig. 3A) although GNA15 is more restricted to neoplastic cells. TACSTD2 is also expressed by epithelial cells ²⁸, particularly squamous, and overexpressed in several adenocarcinoma, particularly squamous cancers (Supplementary Fig. 3B). TACSTD2 over-expression correlates with poor prognosis in pancreatic and other carcinomas, including up to 95% of triple negative breast cancer where Trop-2 represents the target of the FDA-approved antibody-drug conjugate Sacituzumab govitecan (IMMU-132) ²⁸.

We previously evaluated the functional impact of reducing Gα15 expression in the PT45 PDAC cell line. GNA15 was downmodulated by expressing shRNA or completely knocked out by CRISPR/Cas9 ¹⁰. Using the same models, here we analyzed the influence of Gα15 on the abundance of Trop-2 by flow cytometry and western blot. In all cases, Trop-2 expression increased when GNA15 was depleted (Fig. 3A, Supplementary Fig. 4A).

Trop-2 and Gα15 are involved in Ca²⁺ and PKC signaling, carcinoma cells motility ^10,28, PDAC progression and invasiveness ^10,29,30. Several other genes in the signature encode for membrane proteins implicated in PDAC cell motility and reset contacts with the microenvironment (Supplementary Table 2). The GJB3, CDH3, CDCP1 genes encode connexin 31, p-cadherin and CUB Domain Containing Protein 1 (CDCP1) respectively. The latter prevents adhesion through integrin signaling ³¹, is phosphorylated by Src in metastatic cancers and its interaction with PKC contributes to anoikis resistance ³². CDCP1 was identified by a combination of parallel proteomics and CRISPR screenings aimed to identify KRAS induced cell surface proteins ³³.

To explore if GNA15 signature proteins regulate homotypic cell-cell interactions we compared cell aggregation in PT45 clones with or without GNA15. Loss of GNA15 expression increased the ability of cells to form aggregates (Fig. 3B) suggesting Gα15 may promote dispersal of transformed cells.

3.4 GNA15 knockout affects expression of genes instrumental to cell spreading

We next analyzed the impact of GNA15 knock-out (Supplementary Fig. 4B) on the PANC-1 cells transcriptome (Fig. 4A). Gene ontology of RNAseq results showed that cell adhesion and migration were the most significantly impacted by GNA15 depletion (Fig. 4B).

Plasminogen Activator, Urokinase Receptor gene (PLAUR) emerged among the most downmodulated genes, consistent with its role in promoting cell invasion ³⁴. Interestingly, analyzing TCGA-ICGC data for gene expression in neoplasia in various tissues, shows PLAUR is most highly correlated with GNA15, directly followed by the gene encoding its ligand, PLAU. A key role for PLAUR in regulating the shift between single cell tumor dormancy and proliferation has been attributed to PLAUR induction by integrins, resulting in the propagation of signals from fibronectin (FN1) through the EGF-receptor, ERK, and p38 signaling (see Discussion). FN1 was also one of the genes most significantly downregulated by GNA15 knockout in PANC-1 cells. PLAUR and FN1 gene expression levels were validated by TaqMan PCR (Fig. 4c). Additional genes affected by GNA15 knock out support this hypothesis. Of particular interest are the downmodulation of pro-migratory insulin-like growth factor binding proteins (IGFBPs) 1 and 6 ³⁵ and the upregulation of annexin A6 (ANXA6), which prevents migration and invasiveness of squamous epithelial cells ³⁶.

3.5 Gna15 facilitates the development of PDAC precursor lesions in KC mice

Studies in PDAC cell lines showed that KRAS affects the function of membrane proteins to reduce adhesion and promote invasiveness ³³. KRAS mutation is an early event in the etiology of several cancers in humans. Oncogenic KRAS alleles are found in precancerous PanIN-1A (25%) and PanIN-1B (38%) lesions, and predominate in PDAC (85%) ³⁷. Since GNA15 is ectopically expressed by the same lesions ¹⁰, we sought to test if Gα15 contributes to the initiation of pancreatic neoplasia leading to PDAC. A mouse model that expresses oncogenic LSL_Kras^G12D in all cells of the pancreas under control of Pdx1::Cre (KC) from embryonic day 9 (e9) was crossed with a whole body null allele of Gna15 ³⁸ to obtain KC;Gna15^-/- mice (KCG).

Pancreas development and adult function are normal in Gna15 KO mice ³⁸. Analysis was performed on KCG and KC pancreata obtained from either sex at about 40 days of age. At this early stage of initiation, the lesions in both lines of mice are distinct, relatively small, non convoluted and spheroidal ³⁹. Evaluation of serial sections of pancreas from at least 3 different levels separated by 150 µm showed that individual early preneoplastic lesions were morphologically similar but fewer in KCG compared to control KC pancreata (Supplementary Fig. 5 and Supplementary Table 3, p < 0.005). Detailed analysis of a representative H&E section obtained for each mouse quantified all lesions in non-overlapping fields. In total, KC pancreata had a 3-fold greater lesion burden than KCG, displayed higher proportions of fields with single or multiple lesions, and correspondingly fewer negative fields than KCG (61% vs. 86%, Fig. 5).

The majority of KCG lesions occurred individually in well separated fields although two pairs of neighboring fields had two or more lesions (KCG#10 and #12, Fig. 5). By contrast, most lesions in KC pancreata occurred in arrays of 2–6 contiguous fields, and these contained almost all of the fields with more than one lesion. KC had 2-fold higher ratio of fields with one lesion, 8-fold more with two lesions, 10-fold or more with three or four lesions compared to KCG (Supplementary Table 3). Note that nearly every multi-lesion field is neighbored by an empty field in both KC and KCG pancreata, suggesting local signaling drives metaplasia evoked by oncogenic Kras. Because oncogenic Kras is the only driver of neoplasia in the KC model, the results are consistent with the hypothesis that Gα15 ectopic signaling precedes additional driver mutations. Our mouse and human data indicate that regardless of mutations that neutralize oncosuppressors typically driving PDAC, Gα15 signaling intensifies oncogenic KRAS-dependent initiation of preneoplastic lesions in pancreas.

4.1 The appearance of GNA15 signature as an early transcriptional event in PDAC

In the KC mouse model of PDAC, metaplasia appears at 14 days of age and accumulates as the disease progresses from ADM to PanINs ³⁹, providing a sensitive physiologic model for investigating activators and suppressors of oncogenic Kras. Genetic deletion of Gna15 in KC mice significantly reduced precancerous metaplasia at 40 days of age. While Gna15 expression is evidently not required for metaplasia evoked by oncogenic Kras, our mouse and human data indicate that Gα15 signaling intensifies oncogenic KRAS-dependent initiation of preneoplastic lesions in pancreas, even without additional inactivating mutations in oncosuppressors (SMAD4, TP53, CDKN2A) that typically drive PDAC progression. The mechanisms that epigenetically drive GNA15 expression remains to be elucidated but could also control SMAD4 expression levels (Fig. 1) and contribute to intratumoral heterogeneity by determining the multiple clinical and metabolic features associated with molecular subtypes ²⁵. Among 70 genes with the highest Pearson’s scores in the TCGA-PAAD dataset, 7 are located with GNA15 in ch19p.13.3 (Supplementary Fig. 6). This association (p-value = 7.38 e^− 8, FDR q-value = 2.21 e^− 5) could relate to aberrant activation of a transcriptional regulatory program. We previously reported differential methylation of GNA15 promoter as a driver of ectopic Gα15 expression in pancreas ¹⁰. A search for superenhancers (http://sea.edbc.org/) up to 50 kb upstream GNA15 produced 12 hits. A 180 bp long region is marked as an active enhancer by h3k27ac that was previously suggested to influence the expression of PDAC oncogenes ⁴⁰ under KRAS control ⁴¹ and link anabolic glucose metabolism to the formation of distant metastasis ⁴².

Rearrangements in ch19p13.3 were repeatedly associated with a higher incidence of PDAC insurgency ^43–45, indicating a systematic analysis of this region would be valuable. The involvement of several members of the signature in the formation of the trophoblast (Supplementary Table 2) suggests a connection with the changes in protrusive activity, adhesive behavior, and cadherin and connexin expression ⁴⁶. Trop-2 and its homolog EpCAM/Trop-1 were named for being discovered in the epithelial trophectodermal cells that invade uterine epithelial cells ⁴⁷. Placental-cadherin/p-cadherin/CDH3 increased the activity of Rho-family GTPases and consequently cell motility of PANC-1 cells ⁴⁸ and its upregulation favors tumor progression and invasive phenotypes with implications on prognosis ^49,50. We foresee a relationship between the abnormal transcriptional reprograming leading to the expression of the GNA15 signature in PDAC and the transcriptional reprograming leading to placental invasion into the maternal endometrium of the uterus ⁵¹.

4.2 GNA15 signaling and neoplastic cell spreading

GNA15 encodes the Gα15 subunit of the heterotrimeric G protein that couples most GPCRs to the effector protein PLCβ and subsequent Ca²⁺ signaling ^13,52. We previously observed reduced motility and reduced signaling to PKD1 (formerly PKCµ) in GNA15 ko PDAC cell lines ¹⁰. Most genes in the GNA15 signature contribute to form molecular structures that mediate extracellular interactions regulated by Ca²⁺ dynamics ^28,53 and motile phenotype of transformed cells ²⁸ (Fig. 4, Supplementary Table 2). The promiscuous and poorly regulated signaling of Gα15 ¹³ ectopically expressed in early PanIN lesions could cause profound effects in redirecting signaling from a variety of GPCRs implicated in PDAC, such as co-expressed P2RY2 ⁵⁴ and GPR87 ^55,56. Gα15 could cooperate in a macromolecular membrane super-complex similar to the one described to engage Trop-2 and PKC to promote PDAC progression and invasiveness ^29,30. Other intracellular members of the signature are expected to contribute to PDAC invasiveness acting on adhesion molecules (i.e. RhoC regulating α5β ⁵⁷, ANXA6 ³⁶, IGFBP1 and IGFBP6 ^35,58) and proteases (i.e. ubiquitin conjugating enzyme E2C ⁵⁹ and PLAU).

PLAUR and its ligand PLAU emerged as the first 2 genes most highly co-expressed with GNA15 in the pancancer analysis TCGA-ICGC (Supplementary Fig. 7) and the association with GNA15 was confirmed by our data (Fig. 4 and other cohorts). PLAUR encodes for the UPA/PLAU receptor that binds to the GPCR fPRL1. PLAU is strongly implicated in the pathophysiology and clinical outcomes of PDAC patients ⁶⁰ and was recently found to be associated with the metastatic basal-like/squamous and hypoxic PDAC subtype ⁶¹. PLAU and PLAUR can be expressed in response to hypoxic conditions which promote the formation of plasmin, which in turn prompts cell migration. Consistently, PLAUR was prognostic of relapse provoked by tumor cells disseminated in the bone marrow of solid cancer patients ⁶². PDAC patients only occasionally suffer bone metastasis, however, cytokeratin positive cells were reported in bone marrow of 42% of PDAC patients ⁶³. It was speculated that the bone marrow may represent a reservoir of minimal residual disease where tumor cells with low proliferative activity are responsible for relapse of patients with non-metastatic cancer at the time of primary surgery ⁶⁴. We suggest that Gα15 ectopic expression supports the mobilization of transformed slow-dividing cells in the initial phases of the disease and their dissemination to other organs, including in the hematopoietic niche in bone marrow.

4.3 The GNA15 signature suggest that the basal-like/squamous phenotype appears early in PDAC progression

Recent attempts to define combined biomarkers for PDAC prognosis by bioinformatic analysis of existing datasets repeatedly identified GNA15 or other genes in short signatures, alone or combined, as in the case of GNA15 with TACSTD2 ^65–67, PLAUR ⁶⁸, and P2RY2 and LY6E ⁶⁹. GNA15 and its signature represent negative prognostic markers, particularly in PDAC (Fig. 1). This is consistent with the association with invasiveness, the basal-like/squamous phenotype, and more aggressive and advanced stages. However, GNA15 is also involved in PDAC initiation as demonstrated in KCG mice (Fig. 5). Recent phylogenetic studies of human specimens sampled from multiregion metastatic PDAC suggested that cells of ‘classical/progenitor’ and ‘basal-like/squamous’ subtypes co-exist and can transit from one transcriptional type to the other creating intratumoural heterogeneity ^5,25,70. We suggest that the basal-like/squamous and classical/progenitor transcriptional signatures develop sooner than appreciated in humans and the GNA15 signature identifies ‘basal-like/squamous cells’ that propagate early in disease and represent the source of relapse despite surgical treatment. Antibody-drug ²⁸ or antibody-radioisotopes ⁷¹ conjugates have been developed for members of the signature for treatment or diagnostic purposes. A better understanding of the GNA15 signature may provide tools to track elusive PDAC cells and molecular targets to improve treatment.

ADM Acinar to Ductal Metaplasia

Ga15 a subunit of heterotrimeric G15 protein

GNA15 Guanine nucleotide binding protein a15 gene

GPCR G Protein Coupled Receptor

H&E Heamatoxylin and Eosin

IPMN Intraductal Papillary Mucinous Neoplasm

PanIN Pancreatic Intraepithelial Neoplasia

Trop-2 Trophoblastic cell surface antigen 2

PDAC Pancreatic Ductal AenoCarcinoma

PDX Patient Derived Xenograft

PKC Protein Kinase C

Acknowledgments:

This study was performed in the LURM (Laboratorio Universitario di Ricerca Medica) Research Center, University of Verona.

We thank Robert Hammer and the UT Southwestern Transgenic Animal facility for storage and transfer of sperm from Gna15-/- males, Silvano Ross for graphical representation of pancreatic lesions, Robert Hammer, Raymond MacDonald, Rolf Brekken and Alessia Dallatana and our colleagues for comments on the manuscript.

Funding:

AIRC supporting grants IG 17132 and IG 23282 (CB)

NCI grant CA192381 (TMW)

travel award from University of Verona (TMW).

Authors' contributions

Conceptualization: T.M.W., G.M., M.T.V., L.G., C.B., G.I.; Data Curation: T.M.W., Y.Z., R.S., S.P., G.I.; Formal Analysis: S.P.C.; Funding Acquisition: T.M.W., C.B.; Investigation: T.M.W., Y.Z., E.V., M.Z., F.M.; Methodology: Y.Z., G.I.; Resources T.M.W., G.M., R.S., A.P., C.B.; Software: S.PC.; Supervision: Y.Z., , L.G., M.T.V., R.S., S.P., C.B., G.I.; Writing – original draft: T.M.W., G.I.; Writing – review & editing: T.M.W., Y.Z., G.M., L.G., C.B., G.I.

Competing interests

Authors declare that they have no competing interests

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The authors declare no competing interests.

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GNA15 signaling facilitates the initial phases of pancreas cell transformation and is associated with the basal-like/squamous subtype

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Abstract

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1) INTRODUCTION

2) MATERIALS AND METHODS

2.1 Gene expression

2.2 RNAseq differential expression analysis

2.3 Cell Culture

2.4 Western Blot Analysis

2.5 Flow Cytometry

2.6 Cell Aggregation assay

2.7 Mouse model

2.8 Statistical analysis

2.9 Data and materials availability

3) RESULTS

3.1 GNA15 expression in cancer cells is associated with squamous features

3.2 GNA15 expression is associated with an expression signature related to cell-cell interactions

3.4 GNA15 knockout affects expression of genes instrumental to cell spreading

3.5 Gna15 facilitates the development of PDAC precursor lesions in KC mice

4) DISCUSSION

4.1 The appearance of GNA15 signature as an early transcriptional event in PDAC

4.2 GNA15 signaling and neoplastic cell spreading

4.3 The GNA15 signature suggest that the basal-like/squamous phenotype appears early in PDAC progression

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