Pten is a major tumor suppressor in pancreatic ductal adenocarcinoma and regulates an NF-κB-cytokine network

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a dismal 5-year survival rate ranging between 3%–5% (1). Clinically, over 30% of PDAC patients present with advanced primary disease, often with metastases primarily to liver and peritoneum (1). Pathologically, PDAC exhibits a predominantly ductal-like glandular pattern with varying degrees of differentiation that typically arise from precursor ductal lesions termed pancreatic intraepithelial neoplasia (PanIN) (1). A hallmark feature of primary PDAC pathology is desmoplasia characterized by the presence of an exuberant stroma consisting of fibroblasts and inflammatory cells (2). PDAC desmoplasia functions, not only to promote tumor development (3), but also impairs cancer drug delivery by decreasing tumor vascular density and thereby contributing to poor therapeutic response (4). A clear understanding of the pathways and mechanisms shaping the PDAC tumor microenvironment and immune cell infiltration during tumor genesis and progression are not well understood, although paracrine signals involving Tgfβ1 and Hh have been shown to mediate aspects of the pro-tumorigenic communication between tumor cells and surrounding stroma (5-6).

The key genetic alteration driving PDAC genesis is activating KRAS mutations found in greater than 90% of cases and occurs as an early genetic event driving PanIN formation (7-8). While activated KRAS^G12D is the critical event driving PDAC genesis, its precise signaling surrogates and interactions with other signaling pathways remain an area of active investigation. In most tumor cell types, the classical signaling circuits emanating from activated KRAS^G12D include RAF-mitogen-activated kinase (MAPK), phosphoinositide-3-kinase (PI3K), and Ral GDS pathways. However, in human PDAC, mounting genomic evidence has indicated the presence of additional mutations targeting components of the PI3K pathway, suggesting that KRAS^G12D activation of the PI3K pathway is under active repression. Alterations in the PI3K pathway include activating mutations in the catalytic PI3K subunit (PI3KCA), mutations in the regulatory PI3K subunit (PIK3R1/p85α), and amplification of the PI3K downstream effector (AKT2) (9-11). Decreased PTEN expression and accompanying elevations in PI3K/AKT signaling have been observed in pancreatic tumor cell lines, although deletion or loss-of-function mutations targeting PTEN have not been detected with significant frequency in human PDAC (12) and is widely assumed to be a less relevant tumor suppressor in human PDAC. However, previous mouse studies showing that pancreatic deletion of Pten or expression of constitutive active Akt leads to expansion of central acinar cells, putative pancreatic progenitors, and formation of PDAC in a small percentage of mice (13-14), supporting the view that PTEN serves as a major tumor suppressor in murine, and possibly human, PDAC development.

In this study, by using genetically engineered mouse models, we documented strong cooperative interactions of Kras^G12D and Pten loss in promoting metastatic PDAC. Furthermore, molecular analysis indicated that Pten deficient tumors exhibit classic PDAC genetic lesions as well as robust activation of NF-κB and its downstream cytokine pathway, which is associated with inflammatory responses in tumor microenvironment.

RESULTS

The frequent genomic alterations of PI3K pathway components in diverse tumor types prompted analysis of PTEN and AKT status in human PDAC. Tissue microarray (TMA) analysis of PTEN protein and AKT phosphorylation at S473 were measured in a collection of 54 human PDAC samples (Fig.1A). Relative to surrounding stroma, we observed low/no PTEN expression in 70% of PDAC samples (38/54) (Fig.1A-i, ii), findings consistent with previous reports of absent PTEN expression in human PDAC (12, 15). Analysis of phospho-AKT revealed moderate to high AKT phosphorylation in 68.5% of samples (37/54), with most tumors exhibiting moderate levels of AKT activation (30/54) (Fig1A-i). Statistical analysis revealed significant negative correlation between PTEN expression and AKT phosphorylation (p=0.02, Fisher exact test; p=0.03, X² test) (Fig.1A). Correspondingly, high-resolution aCGH analysis of 61 samples of epithelial-enriched primary tumor cells or primary xenografts from PDAC patient samples showed deletion of one or two copies of the PTEN locus in 9/61 (15%) of tumor samples (Fig.1B). PTEN deletion events were typically associated with large regions of 10q chromosome loss, occasionally the whole chromosome arm, - a pattern similar to those in other tumor types (16). In addition, the AKT2 locus was subject to gain/amplification in 12/61 (20%) samples (Fig. 1B), in line with a previous report (11). While only one sample harbored both genetic events, PTEN deletion or AKT2 gain/amplification occurred in 32.8% of PDAC samples (20/61) (Fig.1B). Together, these observations implicate aberrant activation of the PI3K pathway by either PTEN loss or AKT2 activation in a significant subset of human PDACs.

While PI3K is regarded as a major surrogate of KRAS signaling, Pdx1-Cre Kras^G12D PanINs showed a notable lack of robust Akt activation (Fig.S2A-iii). The above genomic data prompted us to hypothesize that PTEN may be actively suppressing the activation of PI3K pathway in Kras^G12D driven lesions. To test this hypothesis, we examined the cooperative interaction of activated Kras^G12D and Pten loss using mice engineered with LSL-Kras^G12D, Pten^L, and pancreatic-specific Pdx1-Cre alleles (17-19). In contrast to Pdx1-Cre LSL-Kras^G12D mice, the Pdx1-Cre LSL-Kras^G12D Pten^L/+ mice (n=40) presented with highly invasive pancreatic tumors and a median survival of 17 weeks (Fig.1C). End-stage histopathological analysis revealed most mice (28/40) with adenocarcinoma exhibiting well-to-moderately differentiated glandular structures which were CK19 positive and devoid of acinar (Amylase) or endocrine (Insulin) markers (Fig.1D). These morphological features are typical of human PDAC (20). In contrast, all 11 Pdx1-Cre LSL-Kras^G12D Pten^L/L mice presented with rapidly progressive acinar-to-ductal metaplasia (ADM) and PanIN formation and exuberant stroma reaction throughout the pancreas with occasional invasive cancers (Fig.S1A). None of these mice survived beyond 3 weeks presumably as a result of severe pancreatic insufficiency. Consistent with previous report (14), Pdx1-Cre Pten^L/L mice progressively develop ADM while Pten heterozygous pancreas is morphologically normal (Fig. S1B).

To examine the impact of single copy Pten loss on evolving pancreatic tumors in the Kras^G12D model, we conducted a detailed serial comparative analysis of Pdx1-Cre LSL-Kras^G12D and Pdx1-Cre LSL-Kras^G12D Pten^L/+ littermates at 4, 6, 8, and 10 weeks of age. Relative to the Pdx1-Cre Kras^G12D model, the phenotype of the Pdx1-Cre Kras^G12D Pten^L/+ pancreas was far more aggressive with significant increase in the number and size of ADM and PanIN lesions (n=5) (Fig.S2B&C), more profound stromal reaction (Fig.S2A-i&v), increased epithelial proliferation (Fig.S2A-iv&viii), and moderately elevated Akt phosphorylation (Fig.S2A-iii&vii). The lesions of both genotypes exhibited similarly strong ERK (Mapk1) activation (Fig.S2A-ii&vi). Together, these data suggest that, in the context of Kras^G12D-initiated pancreatic neoplasms, Pten can repress PI3K signaling and constrain malignant progression in vivo.

One prominent and consistent feature of Pdx1-Cre Kras^G12D Pten^L/+ ductal lesions was a pronounced stromal reaction with inflammatory cell infiltration comparable to that of human PDAC (1). When compared to Pdx1-Cre Kras^G12D littermates, 6 week old Pdx1-Cre Kras^G12D Pten^L/+ animals already exhibited more exuberant reactive stroma surrounding the metaplasia and PanIN lesions which was highly enriched with SMA-positive pancreatic stellate cells and S100A4-positive fibroblasts (Fig.2A-i,ii,iii,vi,vii,viii). In addition, FACS analysis of Pdx1-Cre Kras^G12D Pten^L/+ pancreata showed dramatically increased CD45+ leukocyte infiltration compared to wild-type or Pdx1-Cre Kras^G12D pancreata (Fig.S3). Specifically, in the Pdx1-Cre Kras^G12D Pten^L/+ pancreata, CD11b+Gr-1+ myeloid cells accounted for the majority of the inflammatory cell infiltration, especially Gr-1-low myeloid derived suppressor cells (MDSCs) (Fig.2B), and significant increases in CD25+Foxp3+ regulatory T cells (Tregs) were also documented (Fig.2C). These FACS results aligned with IHC analysis showing obvious myeloid and Treg cell infiltration surrounding metaplastic and PanIN lesions in Pdx1-Cre Kras^G12D Pten^L/+ specimens (Fig.2A-iv,v,ix,x).

Pdx1-Cre LSL-Kras^G12D Pten^L/+ tumors also showed extensive invasion of adjacent organs (Fig.2D). Invasion and metastases into the lymphatic system were also frequently detected (Fig.2D-iii) and multifocal CK19-positive macro- or micro-metastases to liver were observed in 12/35 (34%) of tumor-bearing animals with pathology resembling primary tumors (Fig.2D-iv&v). In contrast, microscopic lung metastasis was rare (1/35) (Fig.2D-vi). Overall, the distribution pattern of liver and regional lymph node metastases is similar to that observed in the human disease (1).

Given the capacity of PTEN to operate as a haploinsufficient tumor suppressor in other settings (21-23), we next examined the status of the remaining wild-type allele of Pten in the Pdx1-Cre LSL-Kras^G12D Pten^L/+ tumors. Early passage cell lines derived from the murine PDACs were used in order to avoid the presence of contaminating normal cells. Allele-specific PCR genotyping revealed that 3/5 tumors retained the wild-type Pten allele as well as Pten protein expression (Fig.3A&B, Fig.S4A), and sequence analysis of the RT-PCR-generated open reading frame (ORF) revealed wild-type Pten sequences (Table S1).

Beyond PTEN analysis, we also audited the status of other key PDAC tumor suppressor genes: Ink4a/Arf, p53, and Smad4. Analysis of the Ink4a/Arf locus revealed loss of p16^Ink4a and p19^Arf expression in all Pdx1-Cre Kras^G12D Pten^L/+ PDAC cells derived from end stage tumors between 11-24 weeks of age (n=5) relative to expression in premalignant pancreatic ductal epithelial cells (PDECs) derived from 6 weeks old Pdx1-Cre Kras^G12D Pten^L/+ or Pdx1-Cre Kras^G12D mice (Fig.3C). The loss of p16^Ink4a expression resulted from gene deletion (1/5) (Fig.S4B, lane 2) or methylation-associated epigenetic silencing (4/5) of the p16^Ink4a promoter region (Fig.S4B, lanes 1,3-5). Examination of the p53 tumor suppressor revealed physical and functional integrity as evidenced by strong induction of p53 and its downstream target p21^CIP1 upon doxorubicin treatment of 5 Pdx1-Cre Kras^G12D Pten^L/+ tumor cell cultures (Fig.3D). All 5 samples tested retained Smad4 expression and sequence analysis of RT-PCR generated Smad4 ORF confirmed the wild-type Smad4 status (Fig.3C; Table S1). Thus, in this model, Pten can function as a haploinsufficient tumor suppressor and extinction of Ink4a/Arf remains a critical cooperative event as classically observed in the human disease (2).

To understand the molecular basis of how oncogenic Kras^G12D and Pten deficiency might cooperate to promote the aforementioned tumor biological features, we performed an unbiased transcriptomic profiling of Pdx1-Cre Kras^G12D PDECs versus Pdx1-Cre Kras^G12D Pten^L/+ PDECs derived from respective 6 week old animals. We employed primary PDECs, as opposed to tumor cells, to better discern the proximal molecular actions of Kras^G12D and Pten loss on premalignant epithelium. The differentially expressed genes (215 genes; fold change ≥1.3; p<0.05) were further analyzed for enrichment of specific binding elements to discern key regulators driving these changes, as reported previously (18). These in silico promoter analyses showed significant enrichment of NF-κB binding elements (Table S2, p=0.03). These observations gain added support in light of NF-κB induction by activated PI3K/AKT pathway (24-25). Accordingly, Pdx1-Cre Kras^G12D Pten^L/+ PDECs exhibited increased nuclear accumulation of NF-κB transcription factor subunit p65 (also known as RelA) (Fig.4A) and higher NF-κB transactivation activity (Fig.4B) relative to Pdx1-Cre Kras^G12D controls. In addition, IHC analysis confirmed markedly elevated levels of phosphorylated nuclear NF-κB in Pdx1-Cre Kras^G12D Pten^L/+ PanINs relative to those morphologically similar lesions in Pdx1-Cre Kras^G12D tissue (Fig.4C), suggesting the activation of NF-κB is unlikely the consequence of different lesion sizes between the two genotypes. Interestingly, although Pdx-Cre Pten^L/L mice develop progressive acinar-to-ductal metaplasia, no significant nuclear accumulation of NF-κB was observed in the ductal lesions (Fig.S5) indicating that both Kras mutation and Pten deficiency are required for activation of NF-κB pathway.

On the functional level, treatment of Pdx1-Cre Kras^G12D Pten^L/+ PDECs with PI3K inhibitor, LY294002 (LY), attenuated nuclear RelA levels and NF-κB transactivation activity (Fig.4A&B) and, conversely, shRNA-mediated knockdown of Pten in Kras^G12D PDECs induced Akt phosphorylation and NF-κB nuclear accumulation (Fig.4D), indicating NF-κB activation is PI3K-dependent. IKK pathway plays essential roles during NF-κB activation (26). However, no obvious induction of IKKα/β activity was observed in Pdx1-Cre Kras^G12D Pten^L/+ PDECs compared to Pdx1-Cre Kras^G12D cells and LY treatment exerted no inhibitory effect on IKKα/β activation as well as the downstream IκBα phosphorylation (Fig.S6). Therefore, our data supports the notion that PI3K-mediated NF-κB nuclear accumulation and activation is likely IKK-independent (24-25, 27). Finally, tumors from Pdx1-Cre Kras^G12D Pten^L/+ mice also showed strong nuclear phospho-RelA signal (Fig.5A). To address the functional relevance of NF-κB activation in these established PDAC tumor cells, we assess tumorigenic activity following introduction of a well-known non-phosphorylatable, dominant-negative mutant of IκBα (IκBαM) (28). As shown in Fig.5B, IκBαM inhibited clonogenic activity of Pdx1-Cre Kras^G12D Pten^L/+ PDAC lines in vitro, indicating cell-autonomous function of NF-κB activation. In addition, suppression of NF-κB also dramatically attenuated tumor xenograft growth in vivo (Fig.5C&D). Together, these data support the view that NF-κB activation plays an important contributory role in the cooperative actions of Kras^G12D activation and Pten deficiency in PDAC development.

Consistent with NF-κB control of cytokine and chemokine expression in numerous cell types including epithelial cells (29), multiple cytokines and chemokines including Il6, Il23, Cxcl1, Ccl20, and Csf3 were found to be significantly up-regulated in Pdx1-Cre Kras^G12D Pten^L/+ PDECs and pancreata relative to controls (Fig.6A, Fig.S7A). Moreover, IκBα mutant expression decreased nuclear NF-κB p65 levels and abolished cytokine and chemokine expression in the Pdx1-Cre Kras^G12D Pten^L/+ PDECs (Fig.6B, Fig.S7B). Consistent with PI3K-directed activation of the NF-κB pathway, PI3K inhibitor, LY294002, decreased cytokine and chemokine levels in Pdx1-Cre Kras^G12D Pten^L/+ PDECs (Fig.6C). Conversely, shRNA-mediated Pten knockdown induced cytokine and chemokine levels in Pdx1-Cre Kras^G12D PDECs (Fig.6D).

These murine observations prompted analysis of the relationship of PTEN status and cytokine and chemokine expression in human PDAC. To that end, we analyzed publically available expression profiles (30) in Oncomine™ and, documented that 4 of the 5 cytokines (CCL20, CXCL1, IL6, IL23) showed significant upregulation in human PDAC compared to normal pancreas and that PTEN expression is significantly downregulated in tumor samples (Fig.S8). Notably, with the exception of CSF3, the upregulated expression of the 4 cytokines showed strong negative correlation with PTEN level (Fig.S8). These studies suggest that the functional genetic interactions between PTEN and the NF-κB-cytokine network appears operative in both mouse and human PDAC, although we acknowledge that multiple genetic mechanisms beyond NF-κB may regulate cytokine expression in human PDAC.

DISCUSSION

In this study, high resolution analysis of human PDAC genomes has uncovered frequent loss of at least one copy of the PTEN gene. In mouse genetic studies, combined genomic, genetic and functional studies establish a key role of the Pten tumor suppressor in the progression of Kras-initiated PDAC development in the mouse. While recent studies have shown Kras and Pten cooperation in mouse PDAC develop (31), our study is distinguished in demonstrating that Pten can function as a haploinsufficient tumor suppressor, aligning well with human genomic profiles, as evidence by retention of Pten heterozygosity in the majority of mouse tumors. Further characterization also established frequent liver metastases. In both mouse and human PDAC, genome scale analysis and confirmatory biochemical studies demonstrate PI3K/Pten-dependent NF-κB pathway activation. NF-κB activation was associated with induction of various cytokines and chemokines which may contribute to the prominent inflammatory cell infiltrates in the Pdx1-Cre Kras^G12D Pten^L/+ tumors. In addition, suppression of NF-κB pathway inhibited Pdx1-Cre Kras^G12D Pten^L/+ tumor growth in vivo, indicating an important role in tumor maintenance hence potential therapeutic target in tumors with altered PI3K regulation.

The lack of Akt activation in Kras^G12D PanIN and metaplasia lesions implied active suppression of the PI3K signaling axis. The activity of PI3K pathway is constantly regulated on many levels involving multiple nodes in feedback loops. This circuitry is in part reflected by coexisting alterations targeting multiple PI3K pathway components in the course of tumor progression, such as the occurrence of KRAS and PTEN mutations in colorectal cancers (32). These observations may align with recent report that p85α, the regulatory subunit of PI3K, directly interacts with PTEN and enhances its phosphatase activity (33). In this context, it is possible that p85α induction of Pten activity in the presence of mutant Kras keeps PI3K signaling in check and that disruption of such regulation by genetic loss/reduction of Pten enables Kras^G12D to drive malignant progression of PDAC. Similarly, ‘reinforcing’ events in PDAC such as mutations of p85α or PIK3CA or amplification of AKT2 would be expected to disrupt negative regulation to ensure the full activation of PI3K signaling in tumorigenesis. The genetic and biological observations of our study justifies the need for a more comprehensive mutational analysis of these components in a large human PDAC sample set and assessment of these profiles with modulation of PI3K signaling and NF-κB network activation.

NF-κB pathway is constitutively active across many different types of tumors including pancreatic cancer (2, 34). NF-κB activation in RAS transformed cells is mediated through both MAPK and PI3K pathways (35). In agreement, our data showed that Pten deficiency cooperates with Kras^G12D to induce NF-κB activity through PI3K pathway. Similarly, NF-κB activity is found to be sustained by elevated PI3K/Akt pathway in several human PDAC cell lines and is important for the viability of the tumor cells (12). Interestingly, PTEN expression itself is suppressed by NF-κB pathway (36). Therefore, PI3K activation via Pten loss and NF-κB may form a powerful positive feedback loop downstream of oncogenic Kras during PDAC development. It was recently shown that loss of p53 also cooperates with Kras^G12D to activate NF-κB pathway in lung adenocarcinoma (37), suggesting that additional mechanisms may also contribute to NF-κB activation in tumors driven by oncogenic Kras. Our data that p53 remains intact in Pdx1-Cre Kras^G12D Pten^L/+ tumor cells and p19^Arf expression is maintained in premalignant ductal cells (Fig.3C&D) suggest that the p53 axis is not prominently involved in the activation of NF-κB pathway in Pten deficient PDECs. At the same time, p53 inactivation through loss of p19^Arf expression in PDAC cells may further contribute to NF-κB activation in the more advanced invasive tumors.

Many in vivo studies have established a central role of NF-κB during tumor development (38). Selective inactivation of IKKβ in either intestinal epithelial cells or myeloid cells suppressed tumor formation in inflammation-induced colorectal carcinoma model, indicating NF-κB pathway functions both cell-autonomously and non cell-autonomously (39). Suppression of NF-κB by dominant negative IκBα mutant inhibits the tumorigenic activity of Pdx1-Cre Kras^G12D Pten^L/+ PDAC cells suggests that NF-κB pathway may function cell-autonomously in established tumors. Meanwhile, our data that NF-κB pathway is activated in Pdx1-Cre Kras^G12D Pten^L/+ PDECs to induce various cytokines and chemokines suggests that NF-κB may also function through paracrine mechanisms during PDAC development to modulate the interaction between tumor cells and its microenvironment, a supposition that will require further study in a defined genetic model.

As an anatomic and functional component of cancer, the composition of tumor microenvironment shows many characteristics observed in chronic inflammation (40). A recent animal study has shown that induction of chronic pancreatitis dramatically accelerated Kras^G12V-induced pancreatic cancer formation, pointing to the importance of permissive environment during Kras-mediated PDAC development (41). However, the underlying mechanisms that orchestrate the cooperation between tumor cells and their inflammatory milieu are less understood. Here we provided additional evidence that the PTEN/PI3K pathway may cooperate with endogenous Kras^G12D to induce the expression of various cytokines and chemokines in pre-malignant epithelial cells in NF-κB-dependant manner. Among the induced cytokines, Il23 and Ccl20 were particularly noteworthy given their well-established roles as chemoattractants for lymphocyte subsets including regulatory T cells (Tregs) (42-43); furthermore, Cxcl1 and Csf3 are integral in the chemotaxis of granulocytes and macrophages to promote tumor progression (44). Such cytokine induction in Kras^G12D Pten^L/+ ductal epithelial cells supported the hypothesis that PI3K activation in combination with oncogenic Kras in epithelial compartment may facilitate the recruitment of various pro-tumor cell types within tumor stroma. The highly enriched cytokines and chemokines in the tumor microenvironment may not only suppress anti-tumor immunity, but the infiltrating inflammatory cells as well as other stromal cells also produce various growth factors to further amplify the PI3K pathway output in Pten deficient cells and support tumor growth. An inbred PDAC model will be critical to further clarify such tumor-microenvironment communication.

Tumors harboring oncogenic Kras mutation are often associated with therapeutic resistance and poor prognosis, and mutant Kras is, until now, still regarded as an ’undruggable’ target (45). Notably, using a Kras mutant animal lung cancer model, a recent in vivo study showed that the combination of a dual PI3K/mTOR inhibitor and a MEK inhibitor induced drastic tumor shrinkage, indicating that targeting Kras surrogates is a feasible therapeutic strategy (46). The strong cooperation between Pten deficiency and oncogenic Kras during PDAC development and the activation of NF-κB pathway under such context further endorses clinical trials for combined therapies with MEK inhibitors and PI3K or NF-κB inhibitors. Alternatively, direct targeting the cytokine networks may diminish immune sequestration in PDAC and enhance immunotherapeutic strategies for this disease.

MATERIALS AND METHODS

Supplementary Material