Targeting acquired oncogenic burden in resilient pancreatic cancer: a novel benefit from marine polyphenols
The upsurge of marine-derived therapeutics for cancer treatment is evident, with many drugs in clinical use and in clinical trials. Seaweeds harbor large amounts of polyphenols and their anti-cancer benefit is linear to their anti-oxidant activity. Our studies identified three superlative anti-cancer seaweed polyphenol drug candidates (SW-PD). We investigated the acquisition of oncogenic burden in radiation-resilient pancreatic cancer (PC) that could drive tumor relapse, and elucidated the efficacy of SW-PD candidates as adjuvants in genetically diverse in vitro systems and a mouse model of radiation-residual disease. QPCR profiling of 88 oncogenes in therapy-resilient PC cells identified a \xe2\x80\x98shared\xe2\x80\x99 activation of 40 oncogenes. SW-PD pretreatment inflicted a significant mitigation of acquired (shared) oncogenic burden, in addition to drug- and cell-line-specific repression signatures. Tissue microarray with IHC of radiation-residual tumors in mice signified acquired cellular localization of key oncoproteins and other critical architects. Conversely, SW-PD treatment inhibited the acquisition of these critical drivers of tumor genesis, dissemination, and evolution. Heightened death of resilient PC cells with SW-PD treatment validated the translation aspects. The results defined the acquisition of oncogenic burden in resilient PC and demonstrated that the marine polyphenols effectively target the acquired oncogenic burden and could serve as adjuvant(s) for PC treatment.
Introduction<!>Materials and methods<!>Xenograft development in mice<!>In vitro and in vivo irradiation procedures<!>Polarity gradient polyphenol(s) extraction and cell treatments<!>Quantitative oncotarget transcriptome profiling<!>Tissue microarray construction, automated immunohistochemistry, and Aperio image analysis<!>Cell death (TUNEL) analysis<!>Cell survival (MTT) analysis<!>Functional relevance of oncotargets in disease progression<!>Acquisition of oncogenic burden in PC cells that survive RT<!>SW-PD treatment prevents oncogenic burden acquisition in PC cells<!>SW-PDs alleviate cellular levels of critical functional oncoproteins in resilient PC<!>SW-PD treatment imposed increased apoptotic cell death in RT-resilient PC<!>Chemical composition of HT-EA extract and anti-PC potential of fractionated products<!>Discussion<!>Conclusions
<p>Pancreatic cancer (PC) remains the deadliest cancer, with an expected 55,440 new cases in 2018 and an estimated 44,330 deaths in the United States [1]. Despite intensive global efforts to improve therapeutic strategies, for more than six decades, PC has had a grim prognosis, with 5-year overall survival (OS) residing around 3–9% [1, 2]. More than half of patients with PC (52%) are diagnosed at a distant stage for which the 5-year OS is only 3% [1]. The current standard of care (SoC) including surgery (only possible in < 20% of patients), radiation therapy (RT), and chemotherapy (e.g., Gemcitabine, Abraxane, Tarceva, and 5-fluorouracil) have together about a billion-dollar US market in this space and still cannot significantly improve PC patient survival. This is mainly attributable to its silent nature, tendency for late discovery [3, 4], presence of occult metastasis at the time of diagnosis, acquired therapy resistance [5–8], and tumor recurrence [9–13]. The use of RT is highly recognized as a neo-adjuvant prior to surgery to shrink/slow PC tumor growth; as an adjuvant after surgery to reduce the chance of tumor recurrence; to shrink/slow the growth of localized, inoperable cancer; and as a palliative to relieve pain [14–16]. Conversely, surviving PC cells at the treatment site after RT may elicit signaling mechanism that may be responsible for clonal selection, tumor cell proliferation/tumor growth, and metastasis [17–19]. Accumulating evidence has demonstrated a tight association between RT and relapse of tumor at the treatment site or at the distant site [20, 21]. Studies have also shown the intrinsic role of oncogene(s) addiction [22–24] and/or activation [25, 26] in tumor relapse, recurrence, and even in the genesis of second cancers. Due to the functional significance of oncogenes in tumor recurrence, it is critical to define and reprogram the acquired oncogene(s) activation, if any, in the cells that survive RT. In the present study, we investigated the oncogene transcriptional rearrangements in PC cells that survive RT and laid out the acquired oncogenic burden in these cells and resilient tumors that could be instrumental in the orchestration of tumor recurrence. Further, we investigated the benefit of novel drug candidates, bioactive polyphenols from seaweeds, in the mitigation of acquired oncogenic burden after RT in PC settings.</p><p>More than 50% of the drugs in clinics, clinical trials, and in the pipeline for cancer treatment are derived and/or designed based on natural products. As an ongoing mission, the National Cancer Institute's (NCI) Development Therapeutic Program (DTP) is screening > 100 k natural product extracts, of which ~ 20 k is marine natural products (MNP) from marine invertebrates, or marine algae (dtp.cancer.gov). Many anti-cancer drugs from marine natural products, including gemcitabine (first-line drug for PC), plitidepsin, glembatumumab vedotin, elisidepsin, PM1004, pseudopterosins, have been promoted to clinical trials and/or even approved by the regulatory agencies [27]. Continuing global efforts in identifying novel superlative anti-cancer drug candidates have recognized significant potential in seaweeds [28]. The wealth of diversity in seaweeds with > 12 K documented species (www.algaebase.org), their availability, edibility, use in traditional medicine, and, more importantly, the high levels of polyphenols (catechin, epicatechin, EGCG, gallic acid) that they harbor [29] brand them as the front-line MNP source. In the context of their anti-cancer properties, recent investigations demonstrated their anti-proliferative [30], tumor-regressing, and metastasis-inhibiting [31] efficacies in many tumor models [32].</p><p>The anti-carcinogenic activity of the seaweed polyphenols is linear and directly proportional to their anti-oxidant activity [33–36]. At the cellular level, studies have shown that the seaweed polyphenols could target cell growth, cell-cycle release, proliferation, survival, DNA repair, angiogenesis, and metastasis, the functional cellular events that drive tumor progression [37, 38]. Recently, we have characterized polarity gradient-based polyphenols from five different marine brown algae, and further branded three superlative candidates on the basis of their anti-oxidant capacity, potential in inhibiting cell viability and proliferation, influencing apoptotic cell death, regulating NFκB transcriptional activity, and targeting critical drivers (kRas, EGFR, AKT, PDGFα, VEGF, TERT, FGF, BCL2, STAT3, AURKβ) of tumor progression in the PC setting [39]. Further, to recognize the benefit of these lead seaweed polyphenol drug candidates (SW-PD) in mitigating the acquired molecular rearrangements in RT-surviving PC cells, those that could drive tumor relapse/recurrence, we sequentially demonstrated their potential in targeting autophagy-mediated activated survival [19], PC cancer stem cell (CSC) clonal selection [18], and heightened metastatic state and potential [17], the three critical functional events of tumor relapse and recurrence.</p><p>In the present study, we investigated the efficacy of SW-PD candidates as adjuvants in mitigating the acquisition of oncogenic burden in radiation-resilient pancreatic cancer (PC), which could drive tumor relapse and recurrence. For this, we used three lead drug candidates: the ethyl acetate fractions of Hormophysa triquerta (HT-EA), Spatoglossum asperum (SA-EA), and Padina tetrastromatica (PT-EA). All of these candidates exhibited high anti-PC potential [39] and demonstrated their individual efficacies in mitigating activated survival, CSC clonal expansion, and metastasis in PC cells that survived RT [17–19]. Recognizing the potential of the SW-PD in the regulation of oncogenic burden in surviving cells could lead to the development/validation of a clinically translatable adjuvant for RT in PC and for possible applications beyond RT and PC.</p><!><p>Human Panc-1 (ATCC-CRL1469), MiaPaCa-2 (ATCC-CRL1420), Panc-3.27 (ATCC-CRL2549), and BxPC-3 (ATCC-CRL1687) cells were cultured and maintained as described earlier [17–19, 39]. For passaging and for all experiments, the cells were detached using 0.25% trypsin/1% EDTA, re-suspended in complete growth medium, counted electronically using a Countess automated cell counter (Carlsbad, CA, USA), and incubated in a 95% air/5% CO2 humidified incubator.</p><!><p>All experiments conformed to American Physiological Society standards for Animal Care, carried out in accordance with the guidelines laid down by the National Research Council, and were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. MiaPaCa-2 cell-line-based xenograft development in athymic nude mice was performed as described earlier [17–19]. At the end of each experiment, animals were euthanized by CO2 asphyxiation, and the xenografts were harvested and subjected to downstream end-point analysis.</p><!><p>For all radiation experiments, PC cells were either mock-irradiated or exposed to fractionated irradiation (FIR) of 2 Gy/day for 5 days at a dose rate of 0.81 Gy/min of low-LET using a Gamma Cell 40 Exactor as described earlier [17–19]. For residual PC studies, PC xenografts developed in nude mice were selectively exposed to clinically relevant FIR (2 Gy/day for 5 days per week for a total of 3 weeks), to a total dose (TD) of 30 Gy. A specially designed cerrobend shield was used to encase the bodies of the mice, exposing only the flank tumors, as described earlier [40–42]. Mock-irradiated animals were treated identically, except they were not subjected to radiation.</p><!><p>Cells were plated in 100-mm tissue culture plates containing 6 ml of complete growth medium, and were allowed to grow up to 70–80% confluence. Polarity-based extractions of seaweed polyphenol fractions were performed as described in our earlier studies [39]. In brief, brown algae, Hormophysa triquerta (HT), Spatoglossum asperum (SA), and Padina tetrastromatica (PT) were collected from the Mandapam coast, Gulf of Mannar, South east coast of India. Freshly collected algae were washed, oven dried and their powder was used for sequential extraction of polyphenols in gradient (increasing) polarity solvents including hexane (H), dichloromethane (DCM), ethyl acetate (EA), and methanol (M). Powdered algae soaked in three volumes of specific solvent were shaken for 48 h at room temperature. The slurry was filtered through Whatman filter paper followed by incubation of the residue in the subsequent solvent. Resulting four filtrates were completely dried in pre-weighed micro-centrifuge tubes using SpeedVac (Thermo scientific, Asheville, NC). The dried filtrates were weighed and dissolved in dimethyl sulfoxide (DMSO) to a final stock concentration of 100 mg/ml. For cell treatment, polyphenol "stock" solutions were further diluted in plain cell culture medium to the desired "working" concentrations. The final concentration of DMSO in the cell culture medium was < 0.0001%. We selectively examined ethyl acetate fractions (HT-EA, SA-EA, PT-EA) that exhibited high anti-cancer activity [39] and mitigated survival, CSC clonal expansion, and the metastatic potential of cells surviving RT [17–19]. In all in vitro investigations, cells were treated with 100 μg/ml of SW-PD, while a corroborated daily (i.v.) dose of 10 mg/Kg concentration was used for in vivo studies.</p><p>Further to characterize the active component(s) within the polyphenol extracts and define the discrete anti-PC benefit of such fractionated compounds, if any, crude polyphenol candidate was fractionated and detected by HPLC/UV. For this purpose, we selected polyphenol candidate HT-EA, which showed relatively higher benefit in this setting. The extract was applied on a C18 column (5 μM, 300A, 0.32 × 150 mm) linked to a HPLC system (Michrom Paradigm MG4) equilibrated with acetonitrile. The column was eluted with a linear gradient of 2% to 90% in the same buffer. The flow rate was maintained at 20 μL/90 s. Fractions were collected and the concentrations were assayed with UV absorption. Altogether, 35 fractions were separated, assayed for intact mass measurement (Ultraflex II MALDI) and the peak characterization and classification was assayed using MZcloud online portal. The separated compounds were further assayed for their potential in inhibiting PC cancer cell survival.</p><!><p>Total RNA extraction and real-time QPCR profiling were performed as described earlier [41], using a custom-made human oncogene transcriptome profiler (Realtimeprimers. com, Elkins Park, PA). The 96-well plate PCR array plates equipped with a set of 88 oncotargets (Table S1) and with eight (GAPD, β-actin, HPRT1, PGK1, RPL13a, GUSb, B2M, and PPIA) housekeeping genes. We custom archived this highly selected QPCR profiler instead of an all-encompassing gene array because the selected genes entail a well-characterized profile governing oncogenesis, disease progression, and tumor evolution, hence facilitating interpretation of data, simplifying data acquisition and analysis, and avoiding genes that are not functionally characterized. More importantly, the archival of this array was primarily designed to focus on disease evolution and, to that end, our bioinformatics analysis with IPA conclusively recognized their high and intrinsic association to pancreatic adenocarcinoma evolution (Figure S1). Furthermore, QPCR profiling allows detection and quantification of gene expression in real-time. The ΔΔct values were calculated by normalizing the gene expression levels to the expression of the housekeeping genes. The normalized data were then compared between groups, and the relative expression level of each gene was expressed as fold change. Group-wise comparisons, heat-map construction, and histogram plots were performed in GraphPad PRISM.</p><!><p>All mouse tissue microarray construction procedures and automated immunohistochemical (IHC) staining were performed in the Stephenson Cancer Center (SCC) - Cancer Tissue pathology core, as described earlier [17–19]. For this study, slides were immunostained for FOSb, cFOS, cMYC, MYB, FRA2, NF2, Col-III, CD73, Cytokeratin wide, Decorin, and Sulf2. Appropriate tissue morphologic/pathologic (H&E) controls and negative controls without primary antibody were examined in parallel. The slides were micro-digitally scanned using an Aperio Scanscope (Aperio Technologies, Inc., Buffalo Grove, IL) slide scanner. Virtual slides were constructed with digital histology. This allows assembly of tissue collections in TMA with variable magnifications. The digital images were then analyzed using Aperio Integrated Spectrum software, the web-based database system. Group-wise comparisons were made using ANOVA with Bonferroni's post hoc correction. A P value of < 0.05 was considered statistically significant.</p><!><p>SW-PD treatment associated increase in programmed cell death was assessed in the SCC-Tissue Pathology Core using a commercially available In Situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) as described in our earlier study [43]. As an appropriate positive control, TMA subjected to induced DNA strand breaks by treating with recombinant DNAse I before TUNEL labeling is included. Corresponding TMA without Tdt enzyme mix were included as negative controls. The slides were micro-digitally scanned using an Aperio Scanscope (Aperio Technologies, Inc., Buffalo Grove, IL, USA) slide scanner. The virtual slide with digital images was then analyzed using Aperio integrated Spectrum (Aperio). TUNEL positivity was quantified using Aperio TMA analysis. Group-wise comparisons were performed using GraphPad Prism.</p><!><p>HPLC/UV-separated fractions (35 fractions in total) of HT-EA SW-PD were examined for their discrete cell killing potential in PC setting. For this, Panc-1 cells were assayed for cell survival using CellTiter 96® AQueous One Solution (Promega) following manufacturer's standard protocols. All assays were performed in duplicate.</p><!><p>First to recognize the functional relevance of the custom archived 88 oncotargets in disease progression, relapse, and recurrence, we plotted their position in terms of cellular location, functional type, participation in signaling events, diseases, and functions, and their driving role in cellular functions. The gene symbols, names, their cellular locations, and their functional types are presented in Table S1. More than half of the archived oncogenes are located in the nucleus (47), while others are located in the plasma membrane (17), cytoplasm (16), and extracellular space (8). Likewise about one half of the archived genes are transcriptional regulators (40) followed by the kinases (23), growth factors (7), enzymes (7), transporters (1), transmembrane receptors (1), G-protein coupled receptors (1), and others (8). Further, characterizing the molecules that were archived in the human oncogene profiler in Ingenuity Pathway (IPA) analysis, we observed that these molecules heavily contribute to the tightly inter-regulated overlapping networks of diseases and function. Notably, IPA analysis identified more than 300 different canonical signaling pathways in which these molecules have critical and functional roles. IPA identified 'Pancreatic adenocarcinoma signaling' as the top canonical pathway with high significance (p 3.89E–29) for the molecules listed here. Furthermore, constructing an overlapping network with the top 50 canonical signaling pathways including the 'direct relationship' and excluding any indirect interactions (Figure S1) revealed a tight and direct association of pancreatic adenocarcinoma signaling with all top 49 critical cancer signaling networks (Figure S1). In addition to their defined roles and association in canonical signaling, we also evaluated their intricate roles in molecular and cellular functions. These 88 oncotargets are critically involved in more than 500 annotated cellular functions, which clearly showed their all-inclusive role in the orchestration of disease progression. For ease of presentation, only the top 25 cellular functions identified by the IPA analysis are included in Table S2. Furthermore, the functional relevance of these archived oncogenes is highlighted by their definitive role in multiple biological functional networks cancer development including cell-cycle, cell death, survival, cellular development, cell growth, proliferation, gene expression cell morphology, post-translational modification, and amino acid metabolism (Figure S2). Together, the IPA analyses signify the functional relevance of the archived 88 oncotargets in cancer progression and hence the activation of such critical drivers in surviving cells after RT (or SoC) could translate to the tumor relapse and recurrence.</p><!><p>To recognize acquired oncogenic burden, if any, we investigated the alterations in the mRNA levels of 88 oncogenes in genetically diverse human PC (Panc-1, Panc-3.27, MiaPaCa-2, and BxPC3) cells that survive fractionated irradiation (FIR, 2 Gy × 5D). The inter-profiler variations normalized QPCR data revealed distinctive gene expression profiles for each treatment and for each cell line investigated (Figure S3). Compared with mock-irradiated cells, the RT-surviving cells showed robust activation of oncogenes in all PC cells investigated (Fig. 1: MiaPaCa-2, 83 genes; Panc-3.27, 79 genes; BxPC-3, 66 genes; Panc-1, 52 genes). Interesting cells → genes traverse analysis identified cell-line-independent activation of 40 genes, including Abl1, Abl2, Akap13, Akt2, Alk, Axl, Bcl2, Bcl3, Bcl6, Brca1, Csf1r, E2f3, Elk1, Erbb2, Erbb3, Erbb4, Fes, Fgf4, Gtf2h1, Hras1, Jund, Kit, Lck, Lyl1, Mcf2, Mlh1, Mll1, Nfkb1, Nfkb2, Ntrk1, Pax5, Pdgfb, Runx1, Stat3, Tgfb1, Tiam1, Tlx1,Tsc2, Vav, and Vegfa across all PC cell lines investigated (Fig. 1, red bars). The relevance of these oncogenes that exhibited cell-line-independent activation after radiation in disease evolution and their association to clinical outcomes are extensively documented in many tumor systems including PC. To that end, our in silico RNA-seq data analysis utilizing R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) in cohorts of 178 (R2-ID tcgapaad178_tcgars), 132 (gse21501), and 96 PC patients (R2 ID nat16965com96_hsens84) clearly recognized the relevance of these candidates in disease progression and poor clinical outcomes. For example, increasing levels of Hras, Pdgfb, Erbb2, Mll1, Tgfb1 and Vav2 showed directly proportional association to advanced disease stages (Figure S4a). Likewise, high expression of Akt2, Axl, Brca1, Gtf2h1, Mll1, Nfkb1, Pdgfb, Vav, or Vegfa (those showed cell-line-independent activation) was inversely correlated with overall patient survival (Figure S4b). In addition, another 28 oncogenes, Junb, June, Mcf2l, Ret, Ros1, Src, E2f1, Egfr, Fgfr2, Fos, Fosb, Gli1, Lmo2, Mas1, Met, Mos, Myb, Myc, Mycn, Nrg1, Pim1, Raf1, Rb1, Stat5b, Tal1, Tal2, Tfdp2, and Tgfb2 showed cell-line-independent expression in at least three PC cell lines investigated (Fig. 1, black bars). Furthermore, 17 oncogenes, including Brca2, Ccnd1, Foxo1, Pms1, Ski, Fosl1, Fosl2, Gip, Tcfl2, Fgr, Hgf, Lyn, Mdm2, Mycl1, Nras, Pdgfa, and Pms2 showed cell-line-independent expression in at least two PC cell lines examined (Fig. 1, blue histograms). Of all the oncogenes activated across cell lines after RT, only one gene, Msh2, showed cell-line-specific (MiaPaCa-2) expression (Fig. 1, blue no fill bar). Such a clear demonstration of cell-line-independent activation of oncogenes in genetically diverse cellular systems illustrates the acquisition of oncogenic burden in PC cells that survive clinically relevant fractionated RT.</p><!><p>Next, to investigate whether SW-PD is beneficial in preventing the acquired activation of oncogenes in RT surviving of PC cells, we examined the levels of oncogenes in cells pretreated with SA-EA, HT-EA, or PT-EA, and subjected to RT. For ease of interpreting the SW-PD-dependent benefit, we performed crisscross analysis between the magnifications of inflicted oncogene activations after RT and SW-PD + RT, and the outcomes are plotted as fold change over RT. SA-EA treatment resulted in the inhibition of 74, 62, 46, and 7 RT activated oncogenes in surviving MiaPaCa-2, Panc-3.27, Panc-1, and BxPC-3 cells, respectively (Fig. 2). Two genes, Bel2 and Runx1, showed cell-line-independent inhibition with SA-EA pretreatment (Fig. 2, red bars). We also observed a cell-line-independent inhibition of 36 oncogenes (Abl1, Abl2, Akap13, Alk, Axl, Bel3, Bel6, Csf1r, E2f3, Elk1, Erbb2, Erbb3, Erbb4, Fes, Fgf4, Jund, Kit, Lck, Lyl1, Mcf2, Mcf2l, Met, Mlh1, Mll1, Nfkb1, Nfkb2, Pax5, Pdgfb, Ret, Src, Stat3, Tgfb1, Tiam1, Tlx1, Tsc2, Vav) in at least three PC cell lines (Fig. 2, yellow bars), and 27 other genes (Akt2, Brca1, Brca2, ccnd1, E2f1, Egfr, Fgfr2, Fgr, Fos, Gtf2h1, Junb, Junc, Lmo2, Mdm2, Myb, Myc, Mycl1, Mycn, Nras, Nrg1, Ntrk1, Pim1, Ros1, Ski, Tal2, Tfdp2, Tgfb2) in two PC cell lines (Fig. 2, blue bars). However, we observed cell-line-specific inhibition of Foxo1 and Hras1 in Panc-1 cells; Gli1, Fosb, Gip, Vegfa, and Hgf in Panc-3,27; and Lyn, Mas1, Mos, Msh2, Pdgfa, Pms1, Pms2, Raf1, Rb1, Stat5b, Tal1, and Tcfl2 in MiaPaCa-2 cells (Fig. 2, unfilled bars).</p><p>Likewise, PT-EA treatment inhibited 70, 57, 45, and 2 oncogenes in MiaPaCa-2, Panc-3.27, Panc-1, and BxPC-3 cells that survived RT. With such a low response rate in BxPC-3 cells, there is no cell-line-independent inhibition across all four cell lines investigated. However, we observed cell-line-independent inhibition of 28 oncogenes, including Abl1, Abl2, Akap13, Akt2, Bcl3, Bcl6, Csf1r, Elk1, Erbb2, Erbb3, Fes, Fgf4, Jund, Lyl1, Mcf2l, Met, Mlh1, Mll1, Nfkb1, Nfkb2, Pax5, Ret, Runx1, Src, Stat3, Tgfb1, Tiam1, and Tlx1 in at least three PC cell lines (Fig. 3, yellow bars). Furthermore, 27 genes (Alk, Axl, Bcl2, Brca1, ccnd1, Erbb4, Fgr, Gtf2h1, Junb, Junc, Lck, Mas1, Mcf2, Mdm2, Myb, Myc, Mycl1, Nras, Nrg1, Ntrk1, Pim1, Rb1, Ski, Stat5b, Tal2, Tfdp2, Mycn) showed cell-line-independent inhibition in two PC cell lines (Fig. 3, blue bars). Conversely, PT-EA exerted a cell-line-specific inhibition of 13 genes (Brca2, Mos, Msh2, Pms2, Raf1, Tal1, Tcfl2, E2f1, Hgf, Lmo2, Lyn, Pdgfa, Tgfb2) in MiaPaCa2; 4 genes (Foxo1, Hras1, Ros1, TSC2) in Panc-1; and 5 genes (Fgfr2, Fos, Fosb, Gli1, Tsc2) in Panc-3.27 cells (Fig. 3, unfilled bars).</p><p>In parallel, HT-EA pretreatment resulted in the inhibition of 82, 67, 36, and 30 oncogenes in radioresistant MiaPaCa-2, Panc-3.27, Panc-1, and BxPC-3 cells, respectively. HT-EA treatment resulted in the cell-line-independent inhibition of Abl1, Erbb4, Fgf4, Gtf2h1, Hras1, Mlh1, Mll1, Pdgfb, and Tlx1 (Fig. 4, red bars). In addition, 35 genes (Akap13, Akt2, Axl, Bcl2, Csf1r, E2f3, Egfr Elk1, Erbb2, Erbb3, Fes, Fgfr2, Fosb, Gli1, Junb, Junc, Jund, Lck, Lyl1, Mcf2, Mcf2l, Met, Mycn, Nfkb2, Nrg1, Pax5, Pim1, Ret, Runx1, Stat3, Tfdp2, Tgfb1, Tiam1, Tsc2, Vav) showed 3 cell-line-independent inhibition (Fig. 4, yellow bars) and another 32 genes (Abl2, Alk, Bcl3, Bcl6, Brca1, ccnd1, E2f1, Fgr, Fos, Foxo1, Hgf, Kit, Lmo2, Mdm2, Myb, Myc, Mycl1, Nfkb1, Nras, Ntrk1, Pdgfa, Pms1, Raf1, Rb1, Ros1, Ski, Src, Tal2, Tgfb2, Vegfa, Fosl2, Gip) showed 2 cell-line-independent inhibition (Fig. 4, blue bars). Conversely, we observed cell-line-specific inhibition of Fosl1, Tcfl2 in BxPC-3 cells, and Lyn, Mas1, Mos, Msh2, Pms2, Stat5b and Tal1 in MiaPaCa-2 cells (Fig. 4, unfilled bars). Together these data clearly demonstrate the potential of SW-PDs in comprehensive inhibition of acquired oncogenic load in RT-surviving PC cells.</p><!><p>Further to substantiate that the acquisition of oncogenic load at the transcript level in RT-resistant PC cells in turn translates to protein expression and/or localization, and to recognize the benefit of SW-PDs in targeting such translation, we investigated the inflicted modulations of critical oncoproteins in RT-resilient PC tissues. For this, we utilized a MiaPaCa-2 cell-derived mouse PC xenograft model. The resilient tumor tissues after clinically relevant RT (2 Gy/Day for 5 days/week for 3 weeks to a total dose of 30 Gy) from animals treated with or without SW-PDs (daily dose of 10 mg/Kg, i.v. at 1 h prior to FIR) were utilized for the TMA construction and examined for modulations in critical oncoproteins using automated IHC. Tumor tissues from mice exposed to mock-irradiation were used as baseline controls. We examined the induced acquisition and SW-PD treatment-dependent regulation of critical oncoproteins Fosb, cFos, Fra2, cMYC, MYB, and NF2. Further, we investigated the key players in tumor evolution, including SELE, CD73, Cytokeratins, Col3, Decorin, and Sulf2. In addition to avoiding any immunostaining inconsistencies across tissues (by TMA construction and automated IHC), we also performed no primary Ab controls for each target to rule out any false positives. Specificity of localization for each target examined was compared for consistency with protein atlas (www.proteinatlas.org). Expression levels across treatment groups were quantified with the Aperio TMA analysis, and only the target localization-specific strong positivity (3 +) was utilized to compute and plot expression as percent resilient PC. Differences between the expression profiles were examined using 2-way ANOVA with Bonferroni's post hoc correction. A P value of < 0.05 was considered significant.</p><p>FBJ murine osteosarcoma viral oncogene human homolog (cFOS, FOS) IHC revealed baseline positivity in mock-irradiated controls. Selective FOS localization was observed in the nucleus and cytoplasm (Fig. 5). We observed strong FOS positivity and abundance in RT-resilient PC tissues (Figs. 5, 6). Though we observed a near identical abundance of FOS reactivity in tissues with SA-EA treatment (Fig. 5), we did not observe any strong positivity (Figs. 5, 6). Likewise, with PT-EA treatment we observed weak cytoplasmic positivity with near-zero nuclear positivity. Localization and expression of nuclear and cytoplasmic FOS was highly restricted with HT-EA treatment (Figs. 5, 6). IHC staining for FosB proto-oncogene, the AP-1 transcription factor subunit, showed a selective strong nuclear localization with weak cytoplasmic localization (Fig. 5). Quantification of nuclear-specific strong positivity across groups revealed a significant increase in the expression of FosB in RT-resilient tumors and was completely regulated with SW-PD treatment. All three SW-PDs investigated showed similar regulation potential of acquired FosB in PC tissues. Staining of another FOS family protein, FOS-like 2 (FOSL2), AP-1 transcription factor subunit (FRA2), revealed cytoplasmic- and nuclear-specific localization (Fig. 5).</p><p>Consistent with FOS, we observed a measurable baseline activity in PC tissues of mock-irradiated animals. Acquisition of FRA2 in RT-resilient PC was evident, with strong nuclear positivity. We observed a significant inhibition of acquired FRA2 localization with SA-EA, PT-EA, and HT-EA treatment (Figs. 5, 6). It is pertinent to mention that all three FOS family proteins examined showed similar expression patterns after RT and similar regulation response with SW-PDs. PC tissues stained for V-myc avian myelocytomatosis viral oncogene homolog (cMYC) showed strong nuclear-specific location and weak cytoplasmic positivity (Fig. 5). Considering only the nuclear-specific localization, expression quantification for definitive, 3+ positivity showed relatively high levels of MYC in PC tissues and served as the positive controls. Expression of cMYC was significantly higher in RT-resilient PC tissues with intense nuclear immunoreactivity. More importantly, SW-PD treatment completely inhibited RT-imposed heightened cMYC localization in resilient PC tissues (Fig. 6). IHC analysis of MYB proto-oncogene, a transcription factor (V-Myb Avian Myeloblastosis Viral Oncogene Homolog), revealed strong nuclear-specific localization (Fig. 5). Compared with mock-irradiated controls, RT resulted in heightened localization of MYB in PC tissues. RT-associated accumulation of MYB in PC tissues was significantly inhibited in the presence of SW-PDs (Fig. 6). Likewise, Neurofibromin 2 (NF2) staining revealed selective cytoplasmic localization in mock-irradiated and in SW-PD-treated groups. Although we did not see any additional nuclear localization in these groups, we observed a strong nuclear localization in RT-resilient PC tissues (Fig. 5). Considering both the cytoplasmic and nuclear localization, expression quantification showed a significant increase of NF2 in RT-resilient PC. This induced increase was completely mitigated with SW-PD treatment (Fig. 6).</p><p>Compared with all protein targets investigated in this study, Endothelial Selectin (SELE) IHC showed near-zero localization in PC tissues and served as the control (Fig. 7). We found a very weak cytoplasm-specific localization of SELE in the tissues. Comparative analysis showed relatively high levels of SELE in RT-resilient tissues, while the treatment with SW-PDs completely reversed this RT-imposed response in PC (Fig. 6). Collagen type III (Col3) staining showed extracellular-specific localization, consistent with its secretory nature (Fig. 7). Compared with mock-irradiated controls, we observed a robust increase in the accumulation of Col3 in RT-resilient tumors. Pretreatment with SW-PDs exerted significant inhibition of the acquired Col3 in PC tissues (Fig. 6). We observed a similar expression profile of 5′-nucleotidase ecto (CD73) in PC tissues. Consistent with the reported localization, IHC staining revealed a selective cytoplasmic and membranous localization of CD73 (Fig. 7). We also noted strong expression of CD73 in mock-irradiated controls. However, compared with mock-IR, we observed a robust increase in CD73 localization. This RT-associated increase in CD73 localization is suppressed in the presence of SW-PDs. We observed the maximal CD73 inhibitory potential with HT-EA treatment (Fig. 6).</p><p>To further investigate the effect of SW-PDs on the RT-modulated cytokeratins, we examined the localization and expression alterations using cytokeratin wide, which covers wide cytokeratin subunits, including 48, 51, 52, 58, 56, and 60 KDa. We observed a strong and selective cytoplasmic and membranous positivity (Fig. 7). Interestingly, we observed a low immunoreactivity in mock-irradiated controls. Compared with mock-IR controls, we observed a significant and intensified CK localization in RT-resilient PC tissues. SA-EA and PT-EA treatment inhibited the accumulation of CK in PC tissues. HT-EA exhibited relatively high efficacy, with complete inhibition of cytokeratins in PC tissues (Fig. 6). Decorin (DCN) staining exhibited selective extracellular localization (Fig. 7). RT-induced extracellular accumulation of DCN was significantly inhibited by SW-PDs. Relatively, HT-EA treatment demonstrated immaculate efficacy in inhibiting acquired DCN in PC tissues (Fig. 6). Interestingly, the SULF2 (Sulfatase 2) staining showed weak positivity in all PC tissues investigated. The localization was specific to the cytoplasm. Though it is a weak positivity overall, the quantification analysis revealed a significant increase after RT and is completely suppressed in the presence of SW-PDs (Fig. 6). Taken together, these data demonstrate the acquisition of critical oncoproteins localization in PC tissues after RT and clearly portraits the benefit of SW-PDs in mitigating induced accumulation.</p><!><p>To underscore the potential of SW-PDs in exerting cell death in RT-resilient PC cells, we investigated the levels of apoptotic cell death in PC tumor tissues after mock-irradiation or FIR with/without SW-PD treatment using TUNEL assay. TMA tissue sections treated with DNase exhibited high levels of TUNEL-positive cells and served as internal positive controls (Fig. 8, top panel). Tdt enzyme controls exhibited zero TUNEL-positive cells and served as the internal negative control. Overall, we observed minimal baseline cell death in untreated mock-IR controls (Fig. 8, lower panel). Quantification of the TUNEL-positive cells in tissues from mock-irradiated animals revealed baseline apoptosis. RT resulted in induced apoptosis, but to a lesser degree. Conversely, treatment with SW-PDs resulted in robust induction of apoptosis in residual tumor (Fig. 8, top panel). We observed an intense induction of apoptotic cell death with HT-EA treatment compared with the other two SW-PDs.</p><!><p>For characterizing the composition of SW-PD, we selected HT-EA extract, which showed relatively better benefit in PC setting. HPLC separation resulted in a total of 35 fractions. However, UV analysis identified three prime fractions (fractions 21, 22, and 23) within HT-EA exhibiting distinctively high concentrations (Figure S5a). Mass spec analysis for all 35 fractions revealed fraction-specific spectrum (Figure S5b). Zooming in for highly concentrated fractions 21, 22, and 23 recognized fraction-specific spectra with unique peaks defying any overlap of neighboring fractions (Figure S5c–e). In particular, fraction 22, with unparalleled concentration in HT-EA, is presented uniquely with one peak, indicating the abundance and probable functional significance of this compound (Figure S5d). Focusing on the observed peaks from fractions 21, 22, and 23, m/zcloud search analysis for peaks (± 1–2 with a resolution power of 1 for m/z accuracy; including reference and autoprocessed libraries and without any compound filter) identified number of related compounds with documented functions including anti-oxidant, anti-cancer, anti-inflammatory (Table S3). More importantly, the single peak observed in fraction 22 primarily corresponded to Salvianolic Acid B. Though the functional significance and bioactive credibility of Salvianolic Acid B have been extensively documented, we acknowledge that further in-depth characterization of this peak (and beyond) is warranted for any such claim.</p><p>Further to define the distinct anti-PC potential of the fractions separated from HT-EA, MTT analysis was performed for all 35 fractions. All fractions exhibited inhibition of cell survival with fraction-specific magnifications (Figure S6). Though most fractions exhibited 20–35% inhibition, we did not see any significant association to the abundance and bioactivity as such.</p><!><p>Dynamic acquisition of genetics and molecular rearrangements in cancer cells are instrumental in coordinating resistance to SoC and tumor evolution [44–46]. As mentioned earlier, PC is mostly diagnosed at later stages [3, 4], and adaptive resistance to current SoC [5–8] leads to poor clinical outcomes [9–13]. Global research efforts are appropriately focused on identifying genetic determinants [47], signal transduction [48], and molecular networks that are involved in PC relapse. Such an approach could be beneficial in developing a new, improved therapeutic strategy for better PC treatment. An altered cellular state that leads to clonal selection and enrichment [6, 49], aberrant survival [48, 50], and pro-metastatic programming is well recognized for prompting PC resistance and relapse [51]. For the first time, the results of the present study described the acquired oncogenic burden in PC cells that survive a course of RT. The outcomes clearly affirmed this response with cell-line-independent activation of 40 oncogenes in genetically diverse PC cells. Furthermore, the activated oncogenes that do not exactly fit cell-line-independent criteria also exhibited shared activation in two or three of the cell lines investigated. Such an immaculate presentation of acquired oncogenic load in PC cells that survive RT portrays the molecular reprogramming that could dictate their aggressive state. To that claim, our IPA analysis recognized the tight association of these activated oncogenes to multiple diseases and functions. This analysis identified pancreatic adenocarcinoma as the top disease that is associated with these oncogenes. Although it could be interesting to discuss the functional relevance of each of these candidates in PC progression, resistance, and disease relapse, such an attempt would only provide an extensive review of the literature here. However, shedding light on their intrinsic roles in adaptive resistance, disease progression, and evolution, our IPA analysis summarized the direct and definitive association of each candidate in canonical signaling, cellular functions, signaling networks, biological functions, and diseases.</p><p>The design and development of novel adjuvants that could target cancer cell adaptive resistance and compliment the current standard-of-care are of great current interest and could shift a major archetype in PC treatment. Strategically, such adjuvants should spare normal tissue toxicity and have a wide spectrum of molecular targeting to address the variations in acquired molecular rearrangements in cancer cells. Molecular rearrangements or genetic determinants could be variable in respect to cell/tumor type, disease stage, genetic make-up, and therapeutic modality. Recent studies have indicated promising benefits from MNPs in many cancer types, including PC [52–55]. More importantly, components (polysaccharides, polyphenols) from marine seaweeds comprise a new class of phytochemicals with unparalleled anti-cancer potential [56–58]. Screening a panel of seaweed polyphenols for anti-cancer abilities, we identified three superlative candidates in the PC setting [39]. Furthermore, we characterized their benefit in targeting (i) the autophagy-driven activated survival of therapy-resistant cells [19]; (ii) the PC-CSCs clonal selection after RT and co-ordination of sternness maintenance [18]; and (iii) the genetic determinant (COX2/CXCR4)-driven dissemination destiny of resilient PC cells [17]. In the present study, for the first time, we identified the adjuvant benefit of SW-PDs in mitigating the acquired oncogenic load in RT-surviving cells. The seaweed polyphenol drug candidates SA-EA, PT-EA, and HT-EA exhibited immense regulatory benefit in the PC setting. Each drug candidate mitigated the RT-imposed oncogenic response across genetically diverse cell lines, demonstrating the efficacy that is necessary to treat heterogeneous PC. Despite holding exceptional anti-cancer benefits, studies that recognize the adjuvant potential of SW-PDs in PC and beyond are highly limited [37, 56, 57, 59–62]. Most of these investigations focused on specific genetic determinant(s) or molecular signaling and did not address disease evolutionary response after SoC. To our knowledge, this is the first study showing the benefit of MNPs, particularly SW polyphenols, in comprehensively regulating the acquired molecular rearrangements, in this case oncogenic burden, after RT. Recognizing their potential in targeting the imposed response across cell lines identifies these compounds as strong, clinically translatable adjuvant candidates.</p><p>Substantiating our oncogene transcript data across PC cell lines, we observed significant accumulation of oncoproteins in residual PC tissues. Our unique RT-residual PC model, coupled with TMA construction and automated IHC, allowed unbiased elucidation of critical oncoproteins and tumor progression drivers/markers. Transcription factors are dysregulated in pancreatic adenocarcinoma. Persistent activation of activator protein-1 (AP-1) is the hallmark of PC. The dimeric complexes of proteins, including c-Jun, JunB, JunD, cFOS, Fra1, Fra2, FosB, and Maf, are denoted as AP-1 with complex regulation of specific gene(s) [63]. cFOS expression has been shown to regulate genes that promote cancer growth, angiogenesis, invasion, and metastasis [64]. The biological effects of cFOS are highly tissue-specific, associated with recurrence, metastasis, and poor prognosis in some cancers (lung, breast cancer, osteosarcoma) [65], and are correlated with favorable survival in others (colorectal, ovarian, and gastric cancers) [66]. However, cFOS has been shown to be highly expressed in PC tissues [67] and plays critical roles in PC initiation and progression. Furthermore, cFOS expression mediates drug resistance through activation of multi-drug resistance gene MDR1, and contributes PC cell motility. Consistently, we observed a robust increase of cFOS localization in RT-residual tumors. In the context of its demonstrated role in PC progression, complete inhibition of cFOS accumulation in residual tumors with SW-PDs defines their benefit. We observed a marked increase of FosB and FRA2 from AP-1 complex, in RT-residual tumors. FOSB proto-oncogene has been shown to drive the expression of critical tumor progression drivers (COX2, VEGF, MMP2, MMP9), to promote cancer cell invasion, and to associate with tumor growth and metastasis [68]. Researchers have reported activated expression of FRA2 in PC cells that survive an insult, and forced expression of FRA2 resulted in tumor(s) formation, including PC, in mice [69]. The results of the present study, for the first time, clearly demonstrate the potential of SW polyphenols in targeting activated AP-1 complex, the dysregulated transcription factor in PC that is instrumental in disease resistance and progression.</p><p>The existence, selection, and/or enrichment of PC-CSCs directly relates to therapy and tumor relapse. In this regard, cMYC is one of the four key transgenes that could reprogram cells into stem cells [70]. The role of MYC in orchestrating tumor cell migration, survival, and invasion has been extensively documented in PC and beyond. More importantly, MYC regulation is critical for acinar maturation and pancreatic homeostasis [71], and MYC expression increases neuroendocrine lineage heterogeneity in PC, prompting disease resistance [72]. Consistent with these studies, we observed a significant increase in MYC expression in RT-resilient PC. The efficient inhibition of activated MYC expression by SW polyphenols observed here critically addresses not only the MYC-dependent PC progression events (survival, invasion, migration) and induced chemoresistance, but also endorses the benefit of SW polyphenols in the regulation of MYC-dependent PC-CSC enrichment and reprogramming of non-CSCs. CD73 (ecto-5′-nucleotidase, ecto-5′-NT), a glycosyl-phosphatidylinositol-linked nucleotidase present in cell membrane lipid rafts, is a defined surface marker of multipotent, slow-growing, self-renewed mesenchymal stem cells. Overexpression of CD73 mediates induced clonal expansion, anchorage-independent spheroid formation, and bidirectional (epithelial/endothelial) differentiation capabilities. In PC, CD73 expression is significantly correlated with tumor invasion and serves as a potential therapeutic target [73]. Our observations of induced CD73 localization in residual tumors after RT go hand-in-hand with these studies. The observed inhibition of CD73 with SW-PDs further affirms the benefit of SW-PDs in targeting PC-CSC enrichment in resilient PC.</p><p>MYB is amplified in PC and showed overexpression in most PC tissues; it signifies as a candidate oncogene [74]. MYB-regulated gene networks and signaling pathways have been documented in PC [75], promote desmoplasia [76], and orchestrate PC growth and metastasis [77]. Consistently, while our findings demonstrated a substantial increase of MYB localization on residual PC, the adjuvant treatment with HT-EA, PT-EA, or SA-EA completely reversed this response. Interestingly, we observed a significant accumulation of NF2 in residual PC that was completely reversed with SW-PD treatment. However, it has been shown that NF2 suppresses PC growth and inhibits metastasis by targeting the FOXM1-mediated Wnt/β-catenin pathway [78]. To that end, the functional relevance of activated NF2 in RT-residual PC is not clear and warrants in-depth investigation.</p><p>Further, we identified that RT resulted in induced SULF2 in residual PC. SULF2 regulates heparan sulfate proteoglycan functions by removing 6-O-sulfation from intact HS-chains, thereby modulating several signaling pathways and leading to carcinogenesis. Studies have linked SULF2 activation to genesis and progression of many cancers [79, 80]. In patients with PC, Sulf2 has been noted as a prognostic biomarker and candidate for therapeutic target [81]. Consistent with our observation of high Sulf2 expression after RT, researchers have documented the involvement of Sulf2 in radiation-induced resistance and invasion of cancer cells [82]. Our results indicated that SW polyphenols effectively inhibited this therapeutic target, and affirm their position as adjuvants.</p><p>In addition, we investigated the induced alterations in matrix proteins decorin and collagen III and the benefit of SW-PDs in reversing the induced response in PC settings. Decorin, a ubiquitously expressed proteoglycan, is expressed in the stroma of a manifold of cancers and is protected from the matrix. Functionally, soluble and matrix-bound decorin modulate several biological processes, including collagen fibrillogenesis, wound healing, stem cell biology, and angiogenesis. In PC, the highest expression of decorin and Col3 has been documented [83]. Although some studies showed that decorin inhibits PC cells growth, recent studies in clinical settings showed that the collagen-associated decorin attenuated the efficiencies of PC chemotherapeutics [84]. We observed a consistent increase of decorin in residual PC and the observed complete reduction of its localization by SW polyphenols could endorse increased sensitization for SoC. Likewise, collagens, particularly type III (COL3), a major constituent of the extracellular matrix, are known to limit the ability of cancer cells to spread. However, high alignment of collagens after PC resection has been shown to be a negative prognostic factor [85]. Thus far, rearrangements of col3, and for that matter any collagen, in PC after RT remain unexplored. To our knowledge, this is the first study showing increased col3 levels in residual tumors. Considering the high alignment of collagens after resection, at present we could only speculate that a similar response after RT may also endorse tumor progression. In light of such claims, the complete suppression of Col3 with SW polyphenols observed in the present study could be beneficial in this setting. Since the influence of SELE in PC cell extravasation is of interest, we investigated the alterations in SELE in residual tumors with and without SW-PD treatment. Interestingly, the staining was weak and skipped beyond any reasonable interpretations in this setting. However, cytokeratins have been employed in diagnostic pathology as markers for several types of human neoplasms, including PC. In the present study, we used a wide spectrum CK to recognize the CK-inhibitory benefit of SW-PDs. Taken together, the results of the present study clearly laid out the molecular rearrangements in residual PC and, for the first time, defined the adjuvant benefit of seaweed polyphenol drug candidates in therapy-resistant PC.</p><p>Also, in this study an attempt was made to characterize the composition of SW-PD, with a special focus to HT-EA extract, which showed better biological benefit. Overall 35 fractions were separated and we identified abundance of three fractions. Mass spec analysis characterized unique spectra and mzcloud peak analysis identified related compounds with documented bio-functions. Our analysis identified that the most abundant fraction (fraction 22) was presented with only one peak, and (with the online portal peak analysis), as such, we could only speculate that this could correspond to Salvianolic Acid B, for which the functional significance and bioactive credibility have been extensively realized. However, it is pertinent to mention that despite the exhibition of cell killing across the fractions examined, the outcomes did not yield any association to the abundance of the fraction and the bioactivity. This could be attributed to the spatial distribution of the bioactive compound across fractions. To that note, we acknowledge that further in-depth characterization of compounds across fractions is warranted. Also, the authors acknowledge the limitations of the present study, including the in vivo toxicity and/or tolerance, bioavailability, and the efficacy validation in clinically translatable transgenic or orthotopic mouse, and the SoC resistance model. Although such research efforts are warranted for the clinical translation of the outcomes and for design and development of adjuvants, it is beyond the scope of this proof-of-concept study.</p><!><p>In conclusion, the outcomes of this study recognized the acquired oncogenic load in RT-surviving cells. Using genetically diverse PC cell lines, we demonstrated that such an acquisition response is not just a random event leading to molecular mayhem. Instead, it is a well-organized process, as evidenced by the cell-line-independent 'shared' activation. Three marine seaweed polyphenol drug candidates (SA-EA, PT-EA, and HT-EA) investigated here strongly mitigated the acquisition of oncogenic load in RT-surviving cells. Each drug candidate inflicted a cell-line-independent inhibition, identifying themselves as promising adjuvants for the treatment of heterogeneous PC. Our unique residual PC model coupled with TMA construction and automated IHC identified the increased localization of critical oncoproteins, including those involved in the AP-1 complex (FOS, FOSb, FRA2), CSC clonal enrichment (cMYC, CD-73), and other disease progression drivers (cytokeratins, Sulf2, decorin, col3, Myb). SW-PD treatment inhibited increased localization of oncoproteins in residual tumors after RT. Further in-depth studies in appropriate preclinical models are warranted and are planned for future investigations.</p>