Tumorigenesis

There is considerable evidence demonstrating the importance of STAT3 in cellular transformation and tumor initiation of PDAC. The hyper-activated state of STAT3 is, in part, due to the presence of oncogenic Kirsten rat sarcoma virus (KRAS) mutations, which account for approximately 90% of PDAC cases [39]. The activation of oncogenic KRAS signaling requires stimulation by upstream factors, and thereby promote aberrant activation of STAT3. In this way, understanding the secretome of the TME is critical to developing novel therapies and improving our understanding of PDAC biology.

As it pertains to cellular transformation and tumorigenesis, STAT3 is critical for the development of pre-malignant pancreatic intraepithelial neoplasia (PanIN) and ultimately progression towards PDAC. This was shown in an elegant study performed by Corcoran et al. [40]. The authors observed increased STAT3 phosphorylation at multiple stages of PDAC development, including the earliest PanIN lesions in KC (Pdx1-Cre; LSL-KRAS^G12D) mouse models [40]. Normal pancreatic tissue, by contrast, had undetectable levels of phosphorylated STAT3 (pSTAT3), suggesting an important role for STAT3 in tumorigenesis and progression. Moreover, is a second study, the authors crossed KC mice with mice containing a conditional knockout (KO) allele for STAT3. STAT3 KO mice exhibited near normal pancreatic histology, demonstrating the critical role of STAT3 in oncogenic KRAS pancreatic cancers [40].

The importance of STAT3 in PDAC tumorigenesis was further confirmed in a study by Loncle et al. [25], where the authors demonstrated that IL-17 induced acinar-to-ductal metaplasia (ADM) and PanIN formation in a STAT3 dependent manner [25]. Specifically, the authors determined that IL-17 induced the expression of regenerating islet-derived protein 3β (REG3β), which in turn promoted cell growth and anti-apoptotic signals [25]. Moreover, REG3 induced the expression of IL-10 and TGF-β via the CXCL12/CXC chemokine receptor 4 (CXCR4) axis, resulting in alternatively activated, or M2 polarized macrophages in co-culture experiments [25]. These data demonstrate that STAT3 contributes to PDAC tumorigenesis and progression, implying a strong rationale for use of STAT3 inhibitors for early intervention strategies of pancreatic cancer.

Proliferation and metastasis

The regulation of multiple key functions within the context of PDAC falls within the functions of STAT3. Both IL-28 receptor, alpha subunit (IL-28RA) and glutathione S-transferase Mu 3 (GSTM3) play a role in the regulation of STAT3 activity, which when overactive, plays a role in cell proliferation, survival, angiogenesis, and metastasis as demonstrated by a reduction of VEGF, when STAT3 is inhibited [6, 41]. Moreover, STAT3, in part, functions as a driver of PDAC growth. When PDAC cells were treated with the EGF receptor (EGFR) inhibitor, AG490, STAT3 activation was inhibited which caused a decrease in cyclin protein expression [6]. The decreased expression indicates that STAT3 plays a critical role in the regulation of the cell cycle and proliferation. In this way, STAT3 serves as a potential therapeutic target, especially in malignancies such as PDAC. Furthermore, using xenograft cancer models in vivo indicated that inhibiting STAT3 with panaxadiol limited the progression of pancreatic cancer [42]. This study also indicates that the use of panaxadiol inhibits cellular processes such as proliferation, migration, and metastasis in Panc-1 and Patu8988 cell lines. Observing the induction of apoptosis in pancreatic cell lines and its strong affinity to STAT3, panaxadiol offers useful insights into the role of STAT3 in cancer proliferation and metastasis through its inhibition.

STAT3 performs important functions, many of which promote an aggressive phenotype for malignant tissues. In normal skin tissues, STAT3 promotes wound healing through cell migration, which cancer cells take advantage of as seen with the migration of ovarian cancer cells [43, 44]. In the context of PDAC, IL-6 and STAT3 each play a role in the promotion of metastasis [8, 9]. Specifically, PSCs secrete IL-6 which activates STAT3 signaling in benign neoplasms and malignant cells, promoting PDAC progression [9]. Inhibition of this crosstalk by using an IL-6 neutralizing antibody, decreased PDAC cell invasion, and colony formation, and STAT3 activation [9]. Certain tumor protein P53 (P53) mutations also promote the migration and invasion of PDAC by promoting the hyperactivation of STAT3 [45]. Mutant P53^R248W protein is stabilized by heat shock protein 90 (HSP90) leading to gain-of-function activity that leads to a hyperactivation state of STAT3, and in turn proliferation and migration. Treatment with HSP90 inhibitors, such as onalespib and ganetespib, causes degradation of mutant P53, thereby reducing STAT3 phosphorylation.

Another method of STAT3 activation relies on the PRL receptor (PRLR) as observed in hormone-dependent cancers, primarily breast cancer, though an observed increase in PRL expression also persists in PDAC [10, 20, 46]. In our previous study, we demonstrated that PRL binds to the extracellular domain of PRLR which promotes an association with JAK2, thus inducing an activation cascade of downstream pathways, such as STAT3 and STAT5 [20]. This increased cellular proliferation, cell motility and stemness of Panc-1 and MiaPaCa-2 PDAC cell lines through STAT3 mediated upregulation of the oncogene, Myc. Moreover, we determined that by inhibiting PRLR induced activation of STAT3 using pharmacologic and genetic approaches, these cellular phenotypes were significantly impaired. As such, PRLR presents an interesting opportunity for the development of therapeutics which target upstream factors of the STAT3 pathway for the treatment of PDAC.

Other health factors influence the function of STAT3 as alternative mechanisms of activation. In PDACs, LDL cholesterol activates JAK1 and JAK2 and non-receptor tyrosine kinases like Src, which promotes cell proliferation and survival by the subsequent activation of STAT3 [29]. Moreover, LDL mediated activation of STAT3 enhances PDAC cell migration and invasion. Treatment of these cancer cells with simvastatin reduced LDL-induced proliferation, cell mobility, and STAT3 activation [18]. This implicates LDL cholesterol in the development and progression of PDAC and indicates that high levels of LDL cholesterol pose an increased risk of PDAC in patients.

Stemness phenotype

There is evidence that a small population of cells within a tumor have an increased capacity to resist therapy and cause tumor recurrence. These cancer stem cells (CSCs) often promote chemoresistance and the recurrence of the disease since they avoid common therapies targeting rapidly dividing cells. These CSCs aren’t necessarily transformed stem cells, but rather malignant cells that have increased ATP-binding cassette (ABC) transporter expression, and DNA repair and reactive oxygen species (ROS) scavenging capabilities [47–49]. Moreover, these CSCs exhibit self-renewal and anchorage independent growth properties [50–53]. As a component of regulating the self-renewal of stem cells, as well as cell survival and inflammation, STAT3 serves a significant role in pancreatic cancer [40]. Recently He et al. [35], determined that IL-22/IL-22RA1 signaling axis contributes to tumor heterogeneity and increased percentage of stemlike cells within the tumor [35]. Accordingly, STAT3 was indispensable for IL-22 induced stemness in pancreatic cancer cells, demonstrating the importance of STAT3 in the pathway. We have also observed that STAT3 enhanced spheroid formation, a surrogate for stemness, in PDAC cells through a PRL: PRLR signaling axis [10]. This may be in part due to increased expression of the STAT3 target gene, Myc. Inhibition of PRLR prevented PRL induced STAT3 phosphorylation and attenuated spheroid formation and induction of stemness genes [10]. As such, STAT3 provides an opportunity for the development of therapeutics that target CSCs.

Metabolism

A key difference between normal and cancer cells is cellular metabolism. As major components in the regulation of cytokine induced survival and proliferation, STAT3 and STAT5 play an important function in tumor growth and survival through the regulation of metabolic processes, such as glycolysis and oxidative phosphorylation [54–57]. In low glucose conditions, nuclear and total STAT3 is decreased, and upon ectopic overexpression of STAT3, cells exhibit increased glucose consumption, indication that aerobic glycolysis is impacted by STAT3 signaling [56]. In another study by Li et al. [55], STAT3 overexpression led to the direct increases in glucose consumption and formation of lactate. This was accompanied by increased expression of hexokinase 2 mRNA and protein, suggesting the direct regulation of glycolytic genes by STAT3 [55]. In the context of PDAC, Valle et al. [57], demonstrated that when galactose was substituted for glucose, PDAC cells utilize oxidative phosphorylation to a greater extent [57]. This caused PDAC cells to become more tumorigenic by enriching for CSC populations and promoting chemoresistance and immune escape [57]. This was associated with noted upregulation in IL-6/STAT3 signaling by ribonucleic acid sequencing (RNA-seq), though validation of the direct role of STAT3 was not tested. Additionally, STAT1 and STAT6 also play a role in metabolism as STAT1, STAT3, STAT5, and STAT6 each play a role in the creation of a dynamic metabolism within cancer cells [58]. STAT3, in particular, supports cancer cell survival in a metabolically hostile environment by promoting an increase in glycolysis through the Warburg effect, which increases proliferation of these cells quickly draining the area around a tumor of vital nutrients and resources [59]. Surrounding cells become starved, while the cells within the tumor continue to thrive, allowing cancer cells to out compete. In this way, the STAT family of proteins provide PDAC tumors with incredible metabolic versatility and adaptability while also influencing other cells within the TME.

Immune cells

STAT3 regulates the communication between cancer cells and immune cells and influences immune escape of tumors [62, 63]. That is, STAT3 drives the immunologically cold state of PDAC tumors. For example, PD-L1 plays an important role in PDAC tumor evasion of the immune system [64]. Under normal circumstances, PD-L1 regulates immune evasion preventing autoimmunity, a function which PDAC tumors exploit [65]. Histone deacetylase 3 (HDAC3) regulates the expression of PD-L1 through the STAT3 pathway as demonstrated by the observed decrease in both PD-L1 and phosphorylated STAT3 in the absence of HDAC3 [66]. This illustrates the importance of STAT3 in the regulation of tumor evasion of the immune system.

The role of STAT3 extends beyond the basic communication between tumors and the TME. Cancer cells require greater resources and nutrients than surrounding cells to sustain their increased proliferation rate. As mentioned earlier, this is regulated in part, by STAT3. The changes in cellular metabolism by cancer cells can also influence the anti-tumor immune responses. The increased nutrient demands by cancer cells can cause oxidative stress on natural killer (NK) cells, which in turn reduces their anti-tumor capabilities [59, 63]. As such, the influence of STAT3 on the TME favors a pro-tumor environment that promotes immune escape. In this way, the manipulation of STAT3 may allow for reactivation of the immune system in patients, thus making STAT3 a potential target in conjunction with immunotherapy. Additionally, the anti-tumor properties of NK cells return upon the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, thus overriding the negative impacts of STAT3 activation on the immune cells [59]. Using this pathway in conjunction with current immunotherapies may allow for the development of improved treatments for PDAC and other cancers.

Fibroblasts

STAT3 also impacts other components of the TME such as the phenotype of fibroblasts, especially those associated with malignant tumors. As such, IL-1 expression in conjunction with the activation of the JAK/STAT pathway, promotes the heterogeneity and inflammation of CAFs which may serve multiple functions [67]. Specifically, the authors determined that two types of fibroblasts exist within PDAC tumors. These two populations of PDAC CAF cells express different markers, critical for their specific functions, with inflammatory CAFs (iCAFs) expressing markers of inflammation such as IL-6 and leukemia inhibitory factor (LIF), and myofibroblastic CAFs (myCAFs) expressing markers of myofibroblasts such as alpha smooth muscle actin (αSMA) [67]. Tumor proximity also segregates these two populations of fibroblast cells, with myCAFs maintaining adjacent positioning with the tumor while the location of iCAFs remains in the stroma further away, though the driving factor for this differential expression remains a mystery [67]. Nevertheless, the secretion of IL-6 by iCAFs can perpetuate the feed-forward loop described above. Interestingly, the authors also demonstrate that TGF-β inhibits IL-1R1 on CAFs, promoting differentiation into a myofibroblasts [67]. This generates a different TME, that is characterized by increased fibrosis. This acts as a double-edged sword, where on one hand the increased fibrosis creates a physical barrier that impairs cancer cell migration, but on the other hand also impedes immune cell infiltration and chemotherapy delivery to the tumor. The authors further demonstrate the role of STAT3, as decreased expression of the iCAF marker genes was observed following STAT3 knockdown [67].

These findings were further supported by a study published by Velez-Delgado et al., where the authors demonstrated that PDAC tumors harboring a KRAS^G12D mutation had increased secretion of inflammatory cytokines, such as CXCL1, IL-6, IL-33, and serum amyloid A (Saa3) that lead to activation of STAT3 in CAFs within the TME [68]. Specifically, the authors utilized a mouse model of pancreatic cancer with inducible and reversible expression of the KRAS^G12D mutation. The authors performed single-cell RNA-seq and mass-cytometry to evaluate the role of mutant KRAS in the TME. These studies demonstrated that oncogenic KRAS signaling within the epithelial tumor cells created an inflammatory secretome that reprogrammed normal fibroblasts towards a tumor promoting phenotype [68].

Collectively, these studies demonstrate that the CAF phenotype in PDAC relies, at least in part, on STAT3 activation. Moreover, the aberrant activation of STAT3 provides direct benefits within the tumor cell itself, and the added benefits of fibroblast reprogramming in the TME to support tumor growth and survival. As such, it will be important to consider the stromal heterogeneity surrounding PDAC for future therapy developments.

Targeting STAT3 in PDAC

The critical role of STAT3 across a broad scope of malignancies has encouraged the development of selective inhibitors for therapeutic and research purposes. Multiple clinical trials continue investigating treatments for PDAC though as the implication of STAT proteins driving the progression of PDAC continues, the need for clinical trials investigating the effectiveness of novel therapies rises. Several clinical trials far have focused on targeting STAT3, yet only two have incorporated pancreatic cancer in the scope of study, to date (Table 2).

A phase II clinical trial with intravenous administration of an anti-sense STAT3 molecule, AZD9150 in combination with an anti-PD-L1 agent, MEDI4736 is currently underway in patients with advanced PDAC. A total of fifty-three patients with either PDAC, as well as those with non-small cell lung cancer (NSCLC), and mismatch repair deficient colorectal cancer. The current anticipated completion date for this trial is April 30, 2024 (NCT02983578). Once complete, this clinical trial may provide unique insight into the feasibility of targeting STAT3 and therefore, warrants revisiting at the time of completion.

The phase III “CanStem111P” clinical trial that was initiated in 2017 was recently terminated due to a lack of OS improvements. This open-label trial across 165 study sites world-wide sought to evaluate the efficacy and safety of combining the STAT3 inhibitor napabucasin with nab-paclitaxel and gemcitabine in patients with metastatic PDAC (NCT02993731). According the Bekaii-Saab et al. [69], 565 patients were randomized into a napabucasin treatment arm and 569 received control (nab-paclitaxel and gemcitabine). Median OS in the napabucasin treatment arm was 11.4 months versus 11.7 months in the control arm. Moreover, no significant difference in OS was observed between biomarker positive subgroups (pSTAT3 positive) [69].

Despite the potential of STAT3 inhibitors in PDAC based on preclinical studies, many trials investigating STAT3 inhibitors in many cancers have failed. This unfortunately creates challenges for evaluating these compounds in pancreatic cancer. One clinical trial studied dosing of OPB-51602, an inhibitor of STAT3 phosphorylation, in solid nasopharyngeal carcinoma tumors. This trial failed due to the intolerable nature of lactic and metabolic acidosis of OPB-51602 (NCT02058017). The consideration of toxic metabolites of a potential drug remains an important factor when determining the viability of such compounds.

Another terminated clinical trial focused on DSP-0337 in patients with solid tumors and ceased activity due to “alternate development strategy” (NCT03416816). Another phase II clinical trial sponsored by the National Cancer Institute investigating the use of flavopiridol in patients suffering from chronic lymphocytic leukemia or prolymphocytic leukemia failed due to serious adverse events (NCT00098371) [70]. While these clinical trials discovered unique issues causing their termination, other trials showed difficulty in recruiting patients. In this way, many clinical trials regarding STAT3 inhibitors in the context of cancer have failed, necessitating the need for new strategies of targeting STAT3 signaling.

Since current STAT3 inhibitors have largely failed in the clinical setting, there is a need to develop novel drugs and strategies to target STAT3. Recently, several small molecule inhibitors have been developed and tested in pre-clinical models of pancreatic cancer. In a study by Chen et al. [71], the authors identified a novel small-molecule inhibitor (N4) that blocked STAT3 phosphorylation by interfering with the SH2 domain. This further prevented STAT3 dimerization and suppressed tumor growth and metastasis in mouse models of pancreatic cancer, providing the justification for the small molecule inhibitor as a potential therapeutic for PDAC [71]. In another study by Zheng et al. [72], the authors tested a novel agent, W2014-S, in NSCLC. This compound worked by not allowing STAT3 to dimerize, and as a result suppressed STAT3 signaling [72]. In animal models of NSCLC, including patient-derived xenograft (PDX) tumor xenografts, W2014-S inhibited cancer growth, demonstrating proof of concept in preclinical studies of STAT3 driven NSCLC tumors [72]. Further evaluation in PDAC may be warranted based on these findings.