Driver Mutations of Pancreatic Cancer Affect Ca2+ Signaling and ATP Production

Abstract Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations—KrasG12D and Trp53R172H—present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since Ca2+ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular Ca2+ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited Ca2+ signals of different frequencies in KPC cells compared to control cells. Finally, Ca2+ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent.


Introduction
Pancreatic cancer (PC) is one of the most lethal malignancies, with an overall survival rate below 11%. 1 Due to the accumulation of g enetic chang es, for example, the presence of "driver mutations" in KRAS and TP53 genes ( Figure 1 ), the exocrine component of the pancreas becomes transformed and loses its functions. 2 , 3 This irr ev ersib l y affects the physiology of the exocrine pancreas, including the production, stor age , and release of digesti v e enzymes.
As counterintuiti v e as it may seem, geneticall y alter ed epithelia of the pancreatic ducts are not the exclusi v e source of cellular precursors for the most common form of PC: pancreatic ductal adenocarcinoma (PDAC). Studies on the phenotypic transition and its role in pancreatic carcinogenesis r ev ealed a considera b le capacity of pancreatic acinar cells (PACs) to undergo morphogenetic changes and, as a result, acquire a duct cell-like phenotype. The consensus has been established that PC arises equally often from the acinar and ductal epithelia. 4 In particular, the multistep cell trans-differentiation processes yield a number of intermediate stages between these two cell types, 5 further contributing to disease pr ogr ession and the intr atumor al phenotypic heterogeneity in PC 6 ( Figure 1 ).
The Kras-dri v en malignant transformation not onl y perpetuates pancreatic carcinogenesis but also induces clonal epithelial loss of function ( Figure 1 ), thus reducing the effecti v e v olume of cells capa b le of hydrolase secretion and production of pancreatic juice. At the early stages of PC development, faulty digestion (often manifesting as diarrhea or constipation) 7 rar el y alerts patients who may simpl y ignor e symptoms of a "clinically silent" disease in the pancreas. However, the pathophysiological mechanisms that dri v e the transition of normal epithelial cells to the premalignant stages of pancreatic carcinogenesis are not well understood.
In normal pancreatic epithelia, the secretion of digestive h ydrolases b y acinar cells occurs via exocytosis concurrent with the secretion of Ca 2 + -rich fluid. [8][9][10] Tightly regulated spatiotempor al oscillations (re gular w av es) of Ca 2 + serv e as a physiological signal for enzyme r elease fr om acinar e pithelial cells. Howev er, when these signals ar e dysr egulated 10 and escalate into global and sustained Ca 2 + elevations, digestive enzymes become activ ated pr ematur el y in situ in PACs, 10 causing necr osis and the release of damage-associated molecular patterns (DAMPs) from injured and dead cells. 11 Surprisingly, the role of pathophysiological Ca 2 + signals is much less evident in the development The image was collected in B-mode, general imaging mode, using the following parameters of signal collection: frequency 40 MHz, power 100%, gain 22 dB, depth 10.00 mm, and width 14.08 mm; and signal display: dynamic range 65 dB, display map G5, brightness 50, and contrast 50. (E) Histological hematoxylin and eosin staining of mouse pancreatic tissues collected from KPC mice at differ ent sta ges of pancreatic carcinogenesis. As the disease progresses, the normal exocrine pancreatic component is lost (in mice > 3-monthold) and becomes gradually replaced by (pre)malignant epithelial structures, and finally with cancerous tissues of 100% organ penetration (mice 6-to 9-monthold). Scale bars: 100 μm.
of PC, compared to other pancreatic pathologies also c har acterized by the inflammatory background, such as acute pancreatitis (AP).
This has prompted us to study cytosolic Ca 2 + signals in PACs isolated fr om KPC (Kras G12D/ + ;Trp53 R172H/ + ;Pdx-1-Cr e) mice, a genetic animal model of pancreatic carcinogenesis. 12 Owing to the presence of mutations in single alleles of the Kras and Trp53 genes under the control of the Pdx1-Cre construct, KPC mice recapitulate a multistep (premalignant to cancer) model of PC dev elopment ( Figur e 1 ). Using KPC and contr ol-Cr e PACs, we first compared Ca 2 + signals elicited by low physiological doses of acetylcholine (ACh) that in normal PACs evoke cytosolic Ca 2 + oscillation. In another set of experiments, we applied menadione (Men) that elicits a mor e irr egular pattern of intracellular Ca 2 + elevations in PACs: oscillations, calcium transients, formation of the cytosolic Ca 2 + plateau, or a combination of all of the a bov e. A detailed numerical analysis of the area under response curves, as well as a direct comparison of the proportions of different types of cytosolic Ca 2 + signal patterns, shows that, on av era ge, Ca 2 + elev ations in KPC cells wer e mor e pr ominent and pr olonged compar ed to contr ols (with Cr e , WT Kr as, and WT Trp53). The application of a Ca 2 + extrusion protocol revealed a significantly slower Ca 2 + efflux from the stores in KPC PACs compared to control-Cre cells, but the expression levels of the plasma membrane Ca 2 + ATPase (PMCA) remained unchanged. Finally, the analysis of the cellular energy metabolism showed decreased mitochondrial respiration in KPC cells compared to normal PACs. Collecti v el y, these findings suggest an impaired Ca 2 + handling in pr emalignant secr etor y e pithelium expr essing Kras G12D and Trp53 R172H , even when no morphological changes are yet evident in PACs.

Isolation of Genomic DNA
5 mm tail tip samples were collected from young animals after w eaning. DN A was extr acted using a Genomic Mini isolation kit, eluted in Tris buffer, and stored for a long term at −20 • C.

Pol ymer ase Chain Reaction
The animal genotype was confirmed by polymerase chain reaction (PCR) using DN A extr acted from the tail tips and specific primers under the conditions described in Ta b le 1 . PCR was carried out in a ProFlex thermal cycler (Thermo Fisher Scientific), in 0.2 mL plastic tube strips containing 3 μL of genomic DNA, 2.63 μL of KAPA, 0.625 μL of 10 μM primer mix, and 6.25 μL of distilled water.

Gel Electrophoresis of PCR Products
For the separation of PCR products (1%: Kras and Pdx1-Cre ; 3%: Trp53 , r especti v el y), 1% or 3% a gar ose gels wer e made by dissolving a gar ose in TAE buffer. Simpl ySafe loading dye w as then added to allow DNA detection in a gar ose gels. Casted gels were positioned in a Sub-Cell GT Horizontal Electr ophor esis System (Bio-Rad, USA), cov er ed with TAE buffer, loaded with 10 μL of DN A or 3 μL of DN A Perfect Leader, and the samples were run at 90 V for 30-60 min. The samples were visualized using a ChemiDoc XRS + Imaging System (Bio-Rad, USA), and the genotypes were determined based on product sizes given in Ta b le 1 .

Experimental Animals
The KPC (LSL-Kras G12D/ + ; LSL-Trp53 R172H/ + ; Pdx1-Cre) 12 mouse breeding pair (mixed background) was transferred from Cardiff Uni v ersity to the J a giellonian Uni v ersity institutional animal units in 2018. The animals were kept in a 12 h light/dark regimen, in indi viduall y v entilated ca ges (up to 5 mice) with aspen wood bedding material and environmental enrichment, and free access to food (standard rodent chow diet) and water. All procedur es inv olving animals wer e performed in accordance with the ARRIVA guidelines, 13 and ultr asonogr aphic tumor detection/palpation was carried out according to license No. 113/2020, issued by the II Local Ethics Committee for Animal Experimentation (Krak ów, Poland). Up to 9-month-old tumor-free KPC mice (males and females, former breeders included), or a ge/sex-matched contr ol animals (LSL-Kras + / + ; LSL-Trp53 + / + ; Pdx1-Cr e; her e, and ther eafter, contr ol-Cr e), wer e humanel y killed by cervical dislocation 14 (for cytosolic Ca 2 + measurements) or CO 2 inhalation (other experiments). The pancreatic tissue was removed for further experimental procedures.

Ultrasound Imaging
In our mouse model of pancreatic carcinogenesis (KPC mice), the presence of pancreatic abnormalities (eg, solid tumors) was examined by abdominal palpation on a weekly basis. In certain cases, KPC mice were also monitored by ultrasound imaging (USG) using a Vevo 2100 high-frequency, high-resolution digital imaging platform (VisualSonics, Canada), according to the previously described protocols. 15 , 16   Excitation was set to 488 nm laser light and emission to 593-630 nm. A series of images were recorded at 256 × 256 pixel resolution; two consecuti v e frames wer e av era ged, and the time resolution was 1 image per 2 s. Fluorescence signals were plotted as F/F 0 , where F 0 was an averaged signal from the first 10 baseline images, normalized as previously described. 20

Calcium Extrusion
For the Ca 2 + -extrusion experiments, PACs wer e pr etr eated with 2 μm thapsigargin (Tg) for 10 min in the absence of extracellular Ca 2 + . Next, again in Ca 2 + -free extracellular buffer, 10 μm Tg was applied for 200 s, and then the extracellular Ca 2 + concentration was increased to 10 m m , which induced Ca 2 + influx to the cytosol. Once a cytosolic plateau w as r eac hed, extr acellular Ca 2 + w as r educed to 0 in the presence of 2 m m EGTA. This abrupt r emov al of extracellular Ca 2 + unmasked Ca 2 + extrusion across the plasma membr ane . This phase of the response was further anal yzed and compar ed between PACs isolated fr om contr ol-Cre and KPC mice. F or e v er y r ecorded Ca 2 + tr ace , the normalized fluorescence that corresponds to half the decrease between the maximum and minimum F / F 0 values was calculated as follows: F 1/2 = F min + ( F max − F min )/2, according to the pr eviousl y described methods. 14 , 21 The time v alues corr esponding to F max ( t max ), and F 1/2 ( t 1/2 ) were calculated from the linear fit to the extrusion phase. Finall y, t 1/2 w as calculated as the difference between t (F 1/2 ) and t max .

Immunoblotting
KPC and contr ol-Cr e mouse pancr eatic tissue samples were homogenized using a TissueLyser (Qiagen, USA), in RIPA buffer supplemented with a protein inhibitor cocktail. The protein concentration was assessed by the BCA assay. 15 μg protein samples were loaded into the NuPAGE 4%-12% Bis-Tris precast gels and r esolv ed (

Cell Death and Viability Measurements
(1) For cell death assessments, PACs wer e stim ulated for 2 h at RT with 60 μm Men (in ethanol stock; or equal volumes of ethanol) in extracellular NaHEPES buffer, containing 1 m m Ca 2 + or Ca 2 +free. One hour before the incubation end, the cells were stained with the NucView 488 & RedDot 2 Apoptosis and Necrosis Kit, which r e ports caspase-3/7 acti vity with a gr een fluor escence signal and the presence of dead cells with far-red fluorescence signal, according to the man ufactur er's instructions. PACs were transferred to a glass-bottom chamber and imaged using a DMI8 fluor escence micr oscope equipped with an HC PL APO 40x/1.30 OIL objecti v e and a DFC7000GT camera (all: Leica, Japan). The following parameters were applied for NucView 488 imaging: excitation 490 nm, 5% illumination power, an FITC emission filter; and for RedDot 2 imaging: excitation 660 nm, 9% illumination power, and a CY5 emission filter. A total of 15 random ima ges wer e collected, and li v e, apoptotic, and necr otic cells were counted, and their numbers were averaged and presented as percentages of the total ± SEM. (2) For the ATP-based cell viability measurements, a CellT iter -Glo 3D Cell Viability Assay was used to quantify ATP levels in acinar cells fr eshl y isolated fr om KPC and contr ol-Cr e mice, following the man ufactur er's pr otocol. Briefly, equal volumes of extracellular NaHEPES buffer containing PACs and CellT iter -Glo 3D Reagent w ere tr ansferred to a 96 Well White/Clear Bottom Plate, shaken for 5 min, and left for 25 min at RT to stabilize the samples. The luminescence signals were recorded using an Infinite M200 Plate Reader (Tecan, Switzerland), and the results were normalized post-assay to the pr otein lev els assessed b y the BCA assa y run in a r efer ence plate, using the same cell aliquot as for the assay.

Cell Metabolism Measurements
Metabolic functions of acinar cells isolated from KPC and control mice were assessed using a Seahorse XF Cell Mito Stress Test Kit, according to the pr eviousl y described pr otocols, 22 , 23 with some modifications. Briefly, 8-well cell culture plates were thin-coated with Matrigel 12 h before the measurements. PACs were isolated on ice, transferred to the plates, and allowed to attach. Ne xt, e xtracellular NaHEPES buffer w as r e placed with XF DMEM pH 7.4 medium, supplemented with 10 m m glucose, 1 m m pyruvate, and 2 m m glutamine. The cells were incubated for 15-30 min at 37 • C in a CO 2 -free I5110 dry oven (Labnet, USA), and then a mitochondrial stress test was performed using a Seahorse Bioscience XF HS Mini Analyzer (Agilent, USA). The cells were injected with sequential fluxes of: 23 1 μg/mL oligomycin applied to inhibit ATP synthase and cause a rapid hyperpolarization of the mitochondrial membranes; 0.3 μm FCCP, an uncoupling agent of oxidative phosphorylation, which restores proton transport across the mitochondrial inner membrane; and 2 μg/mL mix of antimycin A and rotenone, an inhibitor of complexes I and III, and thus a blocker of mitochondrial phosphorylation. The results were normalized post-assay to the cell numbers calculated by counting cell nuclei stained with Hoechst 33258 and analyzed using the QuPath softw ar e. 24 Basal r espiration, ATP production, maximal respiration, proton leak, spare r espirator y capacity, non-mitochondrial oxygen consumption, and coupling efficiency were calculated with normalized oxygen consumption rate (OCR) changes.

Sta tistical Anal ysis
Quantitati v e r esults wer e pr esented as av era ged/r e pr esentati v e linear plots (Ca 2 + traces), box and whisker plots (area under the curve, t 1/2 , relative protein expression, OCR) showing individual data points together with a median and/or a mean, 10 × 10 dot plots (response abundancies), or bar charts (cell death assay) showing a mean ± SEM. For statistical analysis, appropriate parametric or nonparametric tests were applied using the GraphPad Prism 8.0.1 softw ar e (detailed description in the figure legends), and the significance threshold was set at P < .05.

Physiological Ca 2 + Responses in PAC
PACs were isolated from the pancreata of KPC mice (males and females, up to 9-month-old) that carry driver mutations in Kras (G12D) and Trp53 (R172H), under the control of the Pdx1-Cre system, which promotes the development of PC. Control cells were obtained from Cre mice expressing the Pdx1-Cre construct, but in these animals, both alleles of Kras and Trp53 genes were fr ee fr om cancer-inducing point m utations. KPC and contr ol-Cre PACs were isolated by collagenase digestion of the mouse pancreata, and they were essentially identical in terms of their morphological features. In order to c har acterize the capacity of KPC or contr ol-Cr e PACs to maintain physiological Ca 2 + signals e voked b y ACh, we first applied low 50 n m concentrations of the stimulant for 600 s and recorded Ca 2 + responses induced in contr ol-Cr e ( Figur e 2 A) and KPC PACs ( Figur e 2 B). While cytosolic Ca 2 + signals in contr ol-Cr e cells wer e c har acterized by a re gular oscillatory pattern, that is, fine Ca 2 + spikes with regular frequencies and similar amplitudes during the entire ACh stimulation ( Figure 2 A), in KPC cells, these oscillations ev olv ed tow ard more global and sustained Ca 2 + responses ( Figure 2 B). As evidenced by the individual [Ca 2 + ] i traces, not only were KPC acinar cells c har acterized by the initial presence of Ca 2 + spikes at a higher frequency than in controls, but these spikes also had a higher signal amplitude ( Figure 2 A and B). During cell stimulation with 50 n m ACh, Ca 2 + oscillations in KPC cells became graduall y atten uated: both the frequency and amplitudes decreased ( Figure 2 B), but the responses were maintained longer in KPC PACs after r emov al of ACh at 800 s ( Figure 2 A and B). Analysis of the area under the response curve (measured between 200 and 800 s) r ev ealed a significant incr ease ( * P < .05) in the oscillator y r esponses r ecorded in KPC PACs compared to contr ol-Cr e acinar cells (

Cytosolic Ca 2 + Responses to Menadione
Next, we compared the effects of Men on intracellular Ca 2 + signals in KPC and Cre acinar cells. Historically used as a costeffecti v e substitute for vitamin K in fortified foods, Men evokes cytoplasmic Ca 2 + elevations that take the form of oscillatory responses, Ca 2 + transients, or Ca 2 + plateaus (also combinations of all of the a bov e, or no response). Owing to the presence of double bonds in its naphthoquinone ring, Men can initiate the production of reactive oxygen species in PACs. Cell stimulation with 60 μm Men elicited cytosolic Ca 2 + responses in control-Cr e ( Figur e 3 A) and KPC PACs ( Figure 3 B). In both cell phenotypes, the av era ged Ca 2 + tr aces ( F igure 3 A and B) showed the presence of a Ca 2 + peak immediately after Men application, and then the development of differ ent Ca 2 + r esponses, also on top of the elevated Ca 2 + plateau. In KPC cells, Men ev oked intracellular Ca 2 + r esponses immediatel y after the initial peak ( Figure 3 B), while in contr ol-Cr e PACs, the responses w ere delay ed by appr oximatel y 200 s ( Figur e 3 A). This combination of spikes and transients resulted in an irregular pattern of Men-induced Ca 2 + signals, maintained until the application of supramaximal concentrations of ACh, used as controls of the store loading ( Figure 3 A and B). As evidenced by the areas under the response curves to Men and ACh, no statistically significant differences were found between KPC and contr ol-Cr e cells ( Figure 3 C). While oscillations, transients, single spikes, and elev ated Ca 2 + plateaus wer e pr esent both in KPC and in contr ol-Cr e cells, the pr oportions of distinct response types varied between these cells ( Figure 4 A and B). First, as many as 52% of contr ol-Cr e cells and only 22% of KPC PACs did not respond to Men (but responded to ACh). Furthermore, 58% of KPC cells developed one of the following responses c har acterized by the presence of an elevated Ca 2 + plateau: transients on  top of the prolonged plateau (20%), a single spike followed by the plateau (13%), and the plateau alone (25%). The corresponding v alues in contr ol-Cr e cells wer e m uc h low er: 22% of the over all plateau-type responses, that is, 10% of transients on top of the plateau, 4% of a single spike followed by the plateau, and the plateau alone in 8% of total responses recorded in control-Cre cells ( Figure 4 B). In addition, the oscillatory type of responses, Ca 2 + transients, and single Ca 2 + spikes, also showed a different distribution in KPC and contr ol-Cr e cells: 2% vs 4% for the oscillator y r esponses, 15% vs 21% for transients, and 3% vs 1% for single Ca 2 + spikes, r especti v el y. We then compared apop-

Ca 2 + Extrusion Across the Plasma Membrane
In order to compare the speed of Ca 2 + extrusion across the plasma membrane between contr ol-Cr e and KPC cells, we applied a pr eviousl y pub lished pr otocol (with modifications) 14 , 21 ( Figure 5 A). We first incubated PACs in the Ca 2 + -free extracellular buffer supplemented with thapsigargin (Tg), a potent inhibitor of the endomembrane Ca 2 + pump, sarco/endoplasmic reticulum Ca 2 + -ATP ase (SERC A), to empty intracellular Ca 2 + stores. The extr acellular Ca 2 + concentr ation was then c hanged to 10 m m , which caused an influx of Ca 2 + into the cytosol (through the stor e-operated calcium entr y [SOCE])-this w as e videnced b y the increase in cytosolic Ca 2 + until a prolonged plateau was formed in both contr ol-Cr e and KPC cells ( Figure 5 A). Abrupt r emov al of extracellular Ca 2 + and the addition of 2 m m EGTA quickl y r educed cytosolic Ca 2 + concentration to baseline levels, but the apparent rates were different in control-Cre and KPC cells ( Figure 5 A). The continuous presence of Tg in this phase of the experiment blocked SERCA from actively transporting Ca 2 + to the ER. Ther efor e, under conditions of a Ca 2 + -free extracellular environment, the clearance of cytoplasmic Ca 2 + fr om KPC and contr ol-Cr e cells tow ard basal Ca 2 + concentrations reflected Ca 2 + extrusion across the plasma membr ane . Since PACs have only very minor (if any) sodium-calcium exchanger (NCX) activity, 25 almost the entire Ca 2 + extrusion is dependent on the plasma membrane Ca 2 + ATPase (PMCA) in these cells. 21 When extracellular Ca 2 + w as r emov ed, KPC cells not only maintained a Ca 2 + plateau longer than contr ol-Cr e cells but were also c har acterized by slow er r ates of Ca 2 + extrusion than control PACs. The analysis of cytosolic Ca 2 + clearance r ev ealed a statistically significant difference ( * * * * P < .0001) in the PMCA-driven extrusion between KPC and contr ol-Cr e cells, here described as the time r equir ed for Ca 2 + concentration to decrease by half the maximum amplitude (t 1/2 ).

Mitoc hondrial Meta bolism
Aiming to identify the causes of significantly lower Ca 2 + extrusion rates found in KPC acinar cells compared to contr ol-Cr e PACs, we investigated mitochondrial respiration in these cells ( Figure 6 ). While the ATP-based cell viability assay did not show prominent differences between KPC and control-Cre cells ( Figure 6 A), a Seahorse XF Cell Mito Stress Test r ev ealed that the basal respiration and ATP production were decreased (both * P < .05) in KPC cells compared to normal PACs ( Figure 6   Comparison of basal respiration ( * P < .05) and ATP production ( * P < .05) in contr ol-Cr e (orange) and KPC (purple) PACs. Data presented as a box and whisker plot, showing individual data points together with a median and a mean. Statistical significance was assessed using the unpaired t-test, and the P -value < .05 was set as significant. One outlier w as r emov ed fr om the anal ysis of basal respiration (the Grubbs test, α = .05). (D) Comparison of maximal respiration in control-Cre (orange) and KPC (purple) PACs. Data presented as boxes and whiskers, showing individual data points together with a median and a mean. Statistical significance was assessed using the unpaired t -test, and the P -value < .05 was set as significant. (E) 10 × 10 dot plots comparing proton leak, spare respiratory capacity, nonmitochondrial oxygen consumption, and coupling efficiency in PACs normalized to the maximal av era ged v alue measur ed (100%). Gray: differ ence to 100%; color: percentages of the maximal parameter values detected in control-Cre (orange) and KPC (purple) PACs.