A study of 304 human primary pancreatic cancer samples found that 262 (86%) cases were positive for cytoplasmic PAK1 staining, and approximately one-third of all samples showed moderate (2+) to strong (3+) intensity in the malignant cells and nuclear localization of PAK1[57]. Two more recent studies have also shown increased PAK1 expression in resected human pancreatic cancer tissues and cell lines, when compared to the adjacent normal pancreas and an immortalized normal pancreatic ductal epithelial cell line, respectively[58,59]. A PAK1 knock-down pancreatic cancer cell line failed to develop tumours in nude mice[58] and showed markedly reduced proliferation[59]. Furthermore, Jagadeeshan and colleagues demonstrated that fibronectin was a transcriptional target of PAK1 signalling via the NF-κB-p65-fibronectin axis, which modulates cell transformation and the invasive EMT phenotype of pancreatic cancer cells (Figure 3). Early studies also showed the localization of activated PAK1 to the cell nucleus[60] and its involvement in the activation of NF-κB, by demonstrating that active Ras or Rac1 stimulated NF-κB in a PAK1-dependent manner and that active PAK1 itself could stimulate NF-κB as well[61]. An important transmembrane mucin (MUC13) was also reported to be involved in PAK1 signalling in the development of pancreatic cancer[62]. Overexpression of MUC13 promoted cancer cell proliferation, invasion/migration and anchorage-dependent or -independent colony formation in vitro and led to increased xenograft tumour growth and decreased animal survival in vivo. These tumourigenic properties were closely associated with the up-regulated expression and phosphorylation of PAK1, ERK and AKT, and suppression of p53. Wei et al[57] screened a panel of pancreatic cancer cell lines and characterized PAK1 as a key downstream effector of cell motility triggered by multiple growth factors. In their study, PAK1 inhibition not only restored sensitivity to a hepatocyte growth factor/Met antagonist (onartuzumab) in the presence of exogenous growth factors or PAK1-amplification in vitro, but also suppressed tumour growth and metastasis in vivo.

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Figure 3 p21-activated kinases signalling in the development of pancreatic cancer. PAK signalling is involved in several pathobiological processes in pancreatic cancer, including proliferation, migration/invasion, apoptosis and maintenance of stem cell-like properties. Amplification of the PAK1 and PAK4 genes, present within the chromosomal regions 11q13 and 19q13.2, respectively, has been observed. Activated PAK1 regulates cell transformation and the invasive EMT phenotype of pancreatic cancer cells via the NF-κB-p65-fibronectin axis. Additionally, MUC13 promotes cancer cell growth and invasion/migration, and reduces animal survival, by up-regulating expression and phosphorylation of PAK1. Furthermore, PAK4 modulates proliferation and survival by mediating the activity of NF-κB via AKT- and ERK-dependent pathways, and cancer stem cell-like properties via STAT3 signalling. Pharmacological or genetic inhibition of PAK1 or PAK4 leads to decreased cancer cell proliferation, invasion/migration and PSC activation in vitro, and reduced tumour growth and metastasis, and increased animal survival in vivo. PAK: p21-activated kinases; PSC: Pancreatic stellate cells.

In recent years, the interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs) has become the focus of pancreatic cancer research. Activation of PSCs by cancer cells is predominately responsible for fibrosis and stromal remodelling. The role of PAK1 in modulating EMT markers (e.g., fibronectin, E-cadherin, and vimentin) has been established in early studies[63]. A recent study revealed the role of PAK1 in PSC modulation for the first time by showing that inhibition of PAK1 by FRAX597 (a potent group I PAK inhibitor) reduced the activation and proliferation of PSC and increased apoptosis in vitro. In an orthotopic pancreatic cancer mouse model, survival in the PAK1 knockout group was significantly increased compared to the PAK1 wildtype group, and depletion of PAK1 in the pancreatic stroma also reduced PAK1 expression and activity in the tumour[64]. Similarly, survival was prolonged in the group treated with FRAX597 plus gemcitabine in the same orthotopic pancreatic cancer model[59]. These results pave the way to a detailed investigation of the role of PAK1 in tumour-stroma interactions in order to improve therapeutic response by using targeted inhibitors.

PAK4 expression is reported to be correlated with pancreatic cancer pathology. Tyagi et al[65] found 54 out of 56 tumour samples from patients with pancreatic cancer had positive PAK4 staining, with no PAK4 positive staining in normal pancreatic tissue. Furthermore, PAK4 promoted cancer cell proliferation and survival by stimulating the nuclear accumulation and transcriptional activity of NF-κB via AKT- and ERK-dependent pathways (Figure 3). PAK4 knockdown in pancreatic cancer cells caused suppression of growth and colony formation associated with reduced expression of cell-cycle (cyclin A1, D1, E1) and anti-apoptosis (Bcl2, Bcl-xL) proteins. Similarly, inhibition of PAK4 by PF-3758309 (a potent pan-PAK inhibitor) suppressed cancer cell proliferation and migration both in vitro and in vivo[66]. Kimmelman et al[55] identified Rio Kinase 3 and PAK4 as amplified genes in highly recurrent and focal amplifications in pancreatic cancer. Rio Kinase 3 can activate the small GTPase protein Rac, which can subsequently promote cell motility and invasion via PAK4-mediated signalling. In addition, overexpression of activated PAK4 resulted in increased invasion/migration in a gain-of-function experiment, while PAK4 knockdown by shRNA significantly reduced anchorage independent growth in a loss-of-function experiment. Recently, PAK4 has been shown to modulate STAT3 signalling in the maintenance of pancreatic cancer stem cells, which are considered to be responsible for high aggressiveness and chemo-resistance. Pancreatic cancer stem-like cells (CD24⁺/CD44⁺/EpCAM⁺) showed higher PAK4 expression as compared to triple negative cells (CD24^-/CD44^-/EpCAM^-). PAK4 expression enhanced the expression of stem cell-associated transcription factors (Oct4/Nanog/Sox2 and KLF4), whereas PAK4 silencing caused reduced nuclear accumulation and transcriptional activity of STAT3 and loss of stem cell phenotypes[67,68]. The accumulated evidence, which suggests that PAKs are positioned at the convergence point of numerous oncogenic pathways, highlights their potential as promising therapeutic targets in the treatment of pancreatic cancer.

In a prospective randomized clinical study, the human equilibrative nucleoside transporter 1(hENT1), which is the principal cellular transporter of gemcitabine, was found to be a valuable predictive marker of gemcitabine sensitivity in patients with resected pancreatic cancer[78]. In addition, a comparative study also indicated that decreased hENT1 expression was associated with gemcitabine resistance and poorer overall survival in patients with pancreatic cancer[79]. However, an early study reported that up-regulated hENT1 expression was also observed in some gemcitabine-resistant pancreatic cancer cell lines[80]. This evidence implicated hENT1 as the predominant, but not the only, metabolic protein mediating resistance. To some extent, hENT2 and the human concentrative nucleoside transporters hCNT1 and hCNT3 may also contribute to the development of acquired and intrinsic gemcitabine resistance[81,82]. Moon and colleagues have shown that gemcitabine-resistant cell lines express more PAK4 and less hENT1. PAK4 knockdown in gemcitabine-resistant cell lines induces the up-regulation of hENT1 and restoration of sensitivity to gemcitabine[83]. In contrast, one recent retrospective clinical study, which analyzed 160 resected human pancreatic cancer samples by immunohistochemistry, reported that higher expression of PAK4 was correlated with higher expression of hENT1[84]. Therefore, the controversial role of PAK4 in regulating hENT1 should be further explored.

Furthermore, Jagadeeshan et al[85] revealed that PAK1 plays a pivotal role in mediating gemcitabine resistance by altering apoptosis and survival signals, and suppressing DNA damage via the NF-κB pathway. Phosphorylation of PAK1 and ribonucleotide reductase M1 was elevated in patient samples when compared with normal tissue. Combination treatment with a PAK1 inhibitor synergistically improved gemcitabine efficacy and led to tumour regression in animal models. In agreement with this finding, inhibition of PAK1 or/and PAK4 by shRNA knockdown or PAK inhibitors enhanced gemcitabine sensitivity both in vitro and in vivo[59,66].

Other potential PAK-associated signalling pathways also contribute to chemo-resistance. Higher expression of HIF-1α was observed in gemcitabine-resistant pancreatic cancer cell lines[86] and inhibition of HIF-1α can sensitize cancer cells to gemcitabine treatment[87]. The increased activity of HIF-1α was associated with inhibition of the transcription of hENT1 and hENT2, leading to reduced expression of transporter proteins followed by decreased gemcitabine uptake[88,89]. The critical role of PAKs in regulating HIF-1α has been demonstrated in our previous studies, which showed that PAK1 could enhance cancer cell survival by up-regulation of HIF-1α and that inhibition of PAK1 caused decreased expression of HIF-1α and tumour growth[59,90]. Another recent study also found that PAK4 inhibition reduced expression of HIF-1α via the AKT-mTOR-4E-BP1 axis[91].

Additionally, the important transcription factor NF-κB is also a critical regulator closely associated with gemcitabine resistance in pancreatic cancer. As discussed above, localization of active PAK1 to the nucleus is involved in activation of NF-κB[60,61], and both PAK1 and PAK4 contribute to cell transformation, proliferation and survival via NF-κB-dependent signalling pathways in pancreatic cancer[58,65]. Over a decade ago, Arlt and colleagues revealed that resistant cell lines (BxPC-3, Capan-1 and PancTu-1) showed higher expression of NF-κB, comparing to sensitive cell lines (PT45-P1 and T3M4). Treatment of these five pancreatic cancer cell lines with gemcitabine induced NF-κB activity in a dose-dependent manner[92]. Inhibition of the p65 subunit of NF-κB by siRNA can improve gemcitabine sensitivity to suppress proliferation and induce apoptosis both in vitro and in vivo[93]. In agreement with this conclusion, Skrypek et al[94] also showed that decreased activation of NF-κB pathway was correlated with an alteration of hCNT1 expression and increased gemcitabine sensitivity in MUC4-knockdown pancreatic cancer cell lines.

Furthermore, both expression and activity of PAK4 have also been reported to be up-regulated in cisplatin-resistant cancer cells as compared with parental cells. Inhibition of PAK4 diminished cisplatin resistance via PI3K/AKT and MEK/ERK-dependent signalling pathways[95].

The extensive desmoplastic reaction, which is a hallmark of pancreatic cancer, is reported to result in a dense stroma, deficient vascularization and inefficient drug delivery, eventually leading to chemo-resistance[96,97]. As mentioned above, PAK1 is responsible for PSC activation, leading to stromal fibrosis[64] in pancreatic cancer similar to its pivotal role in liver fibrogenic pathways[98].

The importance of Hedgehog (Hh) signalling in tumourigenesis and desmoplasia has been well established. Hh can modify the extracellular matrix component via regulation of the differentiation and motility of PSCs and fibroblasts[99,100]. The observation, in an early global genomic analysis of 24 human pancreatic cancers, that Hh signalling was one of the core set of 12 most commonly altered cellular signalling pathways and was present in 100% of cases suggests a significant contribution of Hh signalling to the development of pancreatic cancer[101]. A number of previous studies also demonstrated that depletion of the Hh signalling pathway could partly diminish desmoplasia-associated resistance and synergistically enhance gemcitabine efficacy both in vitro and in vivo[102-104]. Hiroshi et al[105] found that activation of NF-κB resulted in the aberrant activation of Hh signalling via up-regulation of sonic Hh (a ligand of Hh signalling) in pancreatic cancer.

Interaction of the C-X-C motif chemokine 12 (CXCL12), which is also known as stromal cell-derived factor 1, with its receptor, the C-X-C motif chemokine receptor 4 (CXCR4), can induce activation of downstream signalling pathways related to tumour progression and metastasis[106]. Singh et al[107] have identified the essential role of the CXCL12/CXCR4 axis in stimulation of Hh signalling in a dose- and time-dependent manner. CXCL12-induced Hh up-regulation is due to the increased nuclear accumulation and activation of NF-κB mediated by AKT and ERK signalling pathways. The involvement of PAK1 and PAK4 in NF-κB signalling in pancreatic cancer has been clearly identified[58,61,65]. Although the interaction between PAKs and Hh-mediated chemo-resistance is still not clear, the above findings indicate that PAKs might regulate Hh signalling in a NF-κB-dependent manner and further investigation is needed.

MDSCs, which include both granulocytic and monocytic subsets, are a heterogeneous mixture of activated immature myeloid cells, which can stimulate angiogenesis, promote tumour invasion and migration, and suppress T-cell activation[112]. Both circulating and tumour-infiltrating MDSCs, of the granulocytic subset (Lin-HLA-DR-CD33⁺CD11b⁺CD15⁺), but not the monocytic subset (Lin-HLA-DR-CD14⁺), are markedly elevated in patients with pancreatic cancer compared to the healthy population. Moreover, MDSCs can also serve as an independent prognostic factor for patients’ survival as one unit increase in MDSC percentage leads to a 22% greater risk of mortality[113,114]. The report by Thomas et al[115] of a non-canonical role of the mammalian target of rapamycin (mTOR) protein in recruiting tumourigenic MDSC suggests that cancer cells can stimulate intra-tumoural MDSC accumulation by promoting granulocyte colony-stimulating factor (G-CSF) production via an mTOR-dependent pathway. He and colleagues demonstrated the critical role of PAK4 in regulating mTOR signalling through the PI3K/AKT axis in breast cancer[116]. In addition, PAK1 can be activated by the mTOR/p70 S6 kinase pathway, and treatment with rapamycin, a mTOR inhibitor, leads to reduced PAK1 expression[117]. Interestingly, an early study on vascular smooth muscle cells indicated that G-CSF was involved in activation of the GTPase Rac1, a potent upstream activator of PAK1, and inhibition of Rac1 suppressed G-CSF-driven migration of vascular smooth muscle cells[118]. Previous studies also found that pancreatic cancer cells or stellate cells can attract and transform peripheral blood monocytes into MDSCs via STAT3 activation, which in turn will increase the stem-cell properties and mesenchymal features of tumour cells[119,120]. The role of PAK1 and PAK4 in regulating STAT3 signalling in pancreatic cancer cells has been clearly identified[67,121]. Although there is as yet little direct evidence linking PAK to MDSC modulation, the above findings indicate that PAK might orchestrate multiple signalling pathways to mediate MDSC recruitment and activation.

TAMs can be divided into two subtypes: M1 (pro-inflammatory macrophages) and M2 (anti-inflammatory macrophages). Like MDSCs, the majority of TAMs are derived from circulating monocytes[122]. M1 TAMs can suppress tumour development by stimulating a T-cell-mediated anti-tumour response, whereas the crosstalk of M2 TAMs with tumour and stellate cells can stimulate secretion of various anti-inflammatory cytokines, and reprogram immune surveillance within the tumour microenvironment to facilitate tumour progression[123].

Stephen et al[124] have identified a role of PAK1 in regulating macrophage spreading and lamellipodial dynamics through the activation of ERK1/2. However, they also found that PAK1 knockout had no impact on migration or chemotaxis of macrophages, whereas another study reported that absence of Rac1 or Rac2 could promote macrophage migration[125]. These observations suggest either that PAK2 might compensate for the lack of PAK1, or that other Rac down-stream effectors are involved in regulating cell migration[126]. In addition, Gringel et al[127] found that PAK4 functioned as a physiological regulator of podosomes, which are involved in the migration of human macrophages. Up-regulated expression and activity of PAK4 and its regulator α-PIX (PAK-interacting exchange factor) enhanced the number and size of macrophage podosomes.

There are some additional potential mechanisms linking PAKs to macrophage migration and chemotaxis. The interaction between PAK and HIF-1α has been well established from previous studies[59,90,91]. Recently, HIF-1α was reported to be involved in the recruitment of TAMs in pancreatic cancer through promoting C-C motif chemokine ligand 2 (CCL2) secretion, which stimulated monocyte infiltration into the tumour microenvironment by binding to its receptor CCR2[128]. In agreement with this report, Sanford et al[129] revealed an important role of CCL2/CCR2 in TAM recruitment by showing that a CCR2 antagonist (PF-04136309) was able to block the migration of circulating CCR2⁺ monocytes toward the tumour with a consequent depletion of TAMs in a mouse model of pancreatic cancer. Their clinical data also indicated that pancreatic cancer patients with a higher level of CCL2 expression and greater infiltration of immunosuppressive CCR2⁺ TAMs were significantly more likely to have decreased survival. Additionally, Allen and colleagues revealed the importance of Rho family proteins in regulating actin organization and cell adhesion in macrophages[130]. Using a colony-stimulating factor-1 -dependent murine macrophage cell line (Bac1.2F5), they demonstrated that constitutively activated Rac1 or Cdc42, which are both well-defined up-stream activators of PAKs, could stimulate formation of lamellipodia or filopodia, whereas dominant negative Rac1 or Cdc42 inhibited colony-stimulating factor-1-induced formation of lamellipodia or filopodia.

Macrophage polarization is induced by different stimuli via interferon-regulatory factor/signal transducer and activator of transcription (IRF/STAT) signalling pathways, NF-κB pathways and HIF stabilization. IRF/STAT factors (IRF3, IRF5, STAT1 and STAT5), HIF-1 and the active NF-κB heterodimer (p50-p65) contribute to M1 polarization, while IRF/STAT factors (IRF4, STAT3 and STAT6), HIF-2 and the inhibitory NF-κB heterodimer (p50-p50) trigger an M2 response[131]. The involvement of PAK1 in macrophage polarization has recently been characterized by Zhang and colleagues, who reported that pharmacological or genetic inhibition of PAK1 diminished M1 macrophage polarization. This observation suggested that the up-regulation of PAK1 induced by inflammatory stimuli may contribute to M1 polarization via NF-κB-mediated transcriptional activation. PAK1 was also observed to play a key role in suppressing M2 macrophage polarization[132]. Blockade of the M2 response is an important approach to treatment involving TAM reprogramming. As mentioned above, STAT3 and STAT6 have been reported to be important regulators of M2 polarization. Pharmacological inhibition of STAT3 and STAT6 with specific inhibitors resulted in suppression of M2 polarization, increased production of pro-inflammatory cytokines and stimulated a T cell response[133-135]. Since PAK1 and PAK4 are closely associated with the STAT3 and NF-κB signalling pathway[67,121], it is likely that PAK may interact with STAT3 or NF-κB signalling pathways to block M2 polarization.

Tumour-infiltrating lymphocytes (TILs), including CD4⁺ T cells, CD8⁺ T cells, regulatory T cells, B cells and natural killer cells, are another class of immune cells, which are critical in modulating the tumour microenvironment in pancreatic cancer[136]. Although CD8⁺ T cells are also referred to as cytotoxic T cells, with the capability of recognizing and killing tumour cells, infiltration of CD8⁺ T cells into the tumour microenvironment is very rare. In contrast, a large number of CD4⁺ T cells, which can promote the development of PanINs via inhibition of the anti-tumour response, are observed in the stromal compartment[108,137]. Indeed, an increasing number of studies have revealed the predictive value of stromal TILs in patients with resectable pancreatic cancer. Two of the latest studies have demonstrated that negative stromal TIL patients had larger tumour at a more advanced stage and showed worse overall survival and liver metastasis[138], and that an increased number of tumour-infiltrated CD8⁺ lymphocytes were significantly and independently related to improved disease-free survival and overall survival[139]. Recently, it was reported that pharmacological or genetic depletion of PAK1 up-regulated the immune response to tumours in a colorectal mouse model (APC^14/+ mouse)[140].Similarly, the role of PAK in regulating the tumour-associated immune response was investigated in an murine orthotopic pancreatic cancer model[66]. In agreement with the colorectal model, removal of PAK1 by knock-out or inhibition of PAK1 by PF-3758309 not only suppressed tumour growth in vivo, but also stimulated the immune response by increasing the numbers of splenic CD3⁺ and CD8⁺ T lymphocytes as well as by promoting tumour-infiltrating CD3⁺ T lymphocytes. In contrast, gemcitabine did not significantly change the tumour-associated immune response. Furthermore, it has been reported that granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by tumour cells can recruit and stimulate the development of stromal myeloid cells (Gr-1⁺ CD11b⁺ cells), which can suppress the anti-tumour effect of CD8⁺ T cells[141]. So far, the mechanism by which PAK regulates the production of GM-CSF has not been fully elucidated. However, Kras activation is found to be positively associated with GM-CSF expression in cancer patients when compared to normal controls[142], and this observation is consistent with an early study indicating that oncogenic Kras-dependent secretion of GM-CSF can promote the development of pancreatic neoplasia via immunosuppression mediated by Gr-1⁺ CD11b⁺ myeloid cells[143]. As Kras is the most important oncogenic mutation in pancreatic cancer and a potent up-stream regulator of PAK, these studies provided possible evidence implicating the involvement of PAK in a pathway linking aberrantly activated Kras to GM-CSF-induced immuno-evasion. The mechanisms underlying the connection between PAKs and GM-CSF should be investigated further.