Pancreatic Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

An Introduction to Pancreatic Cancer

Pancreatic cancer is one of the malignant tumors with strong invasiveness, high degree of deterioration and low surgical resection rate in the digestive system, ranking third in cancer-related deaths in the United States. The 5-year survival rate of patients is <5%, and the median survival time is only 5-6 months. According to US cancer statistics, incidence and mortality of pancreatic cancer have continuously increased in recent years. The highly malignant nature of the disease stems from its early local invasion and metastasis. Most patients can be diagnosed by imaging and histopathology, but there are many local or extensive metastases at the time of diagnosis, and no surgical resection can be performed. Therefore, optimizing early diagnosis and developing targeted therapy of pancreatic cancer are the key to improving the survival rate of patients. At present, the key oncogenic pathways of pancreatic cancer including RTK pathway, JNK signaling pathway, TGF-β/SMAD pathway, Wnt signaling pathway, SHH pathway and Notch signaling pathway. These signaling pathways play a very important role in the disease behavior and potential targeting of pancreatic cancer, as well as key proteins and signaling factors in the signaling pathways can serve as potential drug targets. In addition, gene mutations are also crucial in the progression of this malignant tumor. Therefore, validation and optimization of highly sensitive molecular markers can be used not only for earliest stage cancer but also to accurately detect premalignant lesions with high grade dysplasia.

1 Main Signaling Pathways in Pancreatic Cancer Therapy

Fig.1 Pancreatic cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in green) and those in preclinical or early phase development (colored in red) for the treatment of advanced stage pancreatic cancer.

With the rapid development of molecular biology and genomics, it is known that molecular pathogenesis of pancreatic cancer is extremely complicated. Its occurrence, development and metastasis are closely related to a variety of gene mutations and signaling pathways abnormalities, including: K-RAS mutations, epidermal growth factor receptor (EGFR) pathway, pancreatic cancer stem cell signaling pathway, insulin-like growth factor-1 receptor (IGF-1R) pathway, and neovascular dysplasia especially vascular endothelial growth factor pathway. At the same time, these molecular mechanisms provide multiple potential targets for the treatment of pancreatic cancer.

1.1 RTK pathway

Mutation of K-Ras is one of the earliest signs of pancreatic cancer, and it has been reported that up to 90% of patients with pancreatic cancer have detected K-Ras mutations. K-Ras is a small GTPase that acts to induce downstream signaling pathway receptor tyrosine kinases such as EGFR. K-Ras oncogene mutation its induction function and activation of the extracellular signal-regulated kinase pathway (MAPK; ERKs). Once activated, ERK is transferred to the nucleus and promotes the transcriptional activity of target genes, including cell survival, growth and proliferation. Ras oncogene also interacts with other essential signaling molecules. Phosphatidylinositol-3-OH kinase (PIK3CA) is an important signal transduction molecule for protein synthesis at G1 phase of cell cycle and was shown to be a target for the Ras gene in the early 1990s. PIK3CA, activated by K-Ras and other growth-stimulating receptors (such as EGFR), promotes tumor cell growth and causes cells to continue into the next phage of growth cycle. In addition, the K-Ras gene can also interact and activate c-Jun N-terminal kinase (JNK) and protein kinase C (PKC), thereby promoting cell growth from nother pathways. The series ofsignaling pathway of K-Ras indicates that signaling network in pancreatic cancer cells is extremely complex. Once the survival pathway associated with growth stimulation of cancer cell is activated, other cellular responses will also be initiated to cope with the cellular stress and changes in the microenvironment. At the same time, K-Ras can actively turn on the cell reprogramming process, thereby inducing the transformation of acinar cells into malignant clonal cells.

The epithelial growth factor receptor (EGFR, also called HER) family is a subset of receptor tyrosine kinases that have been shown to be active in many epithelial cancers such as colorectal cancer, breast cancer and lung cancer. It is an activator of a series of signaling pathways and other growth factors, such as K-RAS, PIK3CA, Signal Transduction and Transcriptional Activator (STAT), and phospholipase C (PLC). Promoted by these pathways, cancer cells can be modified in gene expression profiles and vested with unique features in cell growth, proliferation and invasion. At present, the phenomenon of EGFR overexpression in pancreatic cancer tissues has been demonstrated. Up-regulation expression of EGF and overexpression of EGFR suggest that there may be a closed loop in regulation between the receptor and the ligand. Mutations affecting RTKs, Ras, B-Raf, PI3K and AKT are common in perpetuating the malignancy of pancreatic cancer.

1.2 JNK signaling pathway

c-Jun N-terminal kinases (JNKs) belong to the family of mitogen-activated protein kinases (MAPKs) and are involved in a wide spectrum of cellular processes, including cell proliferation, differentiation, migration, inflammation, and apoptosis. JNK pathway is activated by two upstream mitogen-activated protein kinase kinases (MAP2Ks) (MKK4 and MKK7) that directly phosphorylate JNKs on Thr183 and Tyr185 residues. In turn, MKK4 and MKK7 are activated by upstream pathways through various mitogen-activated protein kinase kinase kinases (MAP3Ks), including MEKK1-4, MLK2 and -3, Tpl-2, DLK, TAO1 and 2, TAK1, and ASK1/2, which could be specific to different stimuli. A large number of nuclear proteins, mainly transcription factors and nuclear hormone receptors which regulate a plethora of cellular activities, such as proliferation, differentiation, survival, and apoptosis have been identified as JNK substrate proteins. Jun proteins, particularly c-jun are the most important nuclear substrates, which, once activated, form the transcription factor activator protein-1 (AP-1) after dimerization with Fos proteins. Other JNK downstream signaling molecules include activating transcription factor 2 (ATF-2), c-Myc, p53, Elk1, nuclear factor of activated T cell (NFAT), signal transducers and activators of transcription (STAT)1 and 3, and the family protein Pax, as well as mitochondrial apoptosis regulators of Bcl-2 family. Complexity of JNK signaling plays significant role in normal cellular functions including controlled degradation of proteins, cell and tissue morphogenesis, and immune response. However, More recent data suggested that JNKs and especially JNK1 contribute to malignant transformation and tumor growth, and it is widely known that malignant transformation induced by Ras oncogene is regulated by c-Jun. In addition, several studies demonstrated that upregulation of JNK enhances tumor growth or induces drug resistance in pancreatic Cancer.

1.3 TGF-β/SMAD signaling pathway

TGF-β pathway is a very complex signaling network, and its role in cellular homeostasis varies with different genetic profiles of cancer cells. On the one hand, TGF-β signaling can induce cell differentiation and act as a tumor suppressor in non-malignant tumor, on the other hand, it is also capable of promoting angiogenesis and EMT in cancer cells. Once signal transduction is initiated, TGF-β is involved in cell differentiation and can also act as a tumor suppressor in cell cycle control. Furthermore, TGF-β induces p53-independent expression of p21 and inhibits oncogene expression, while TGF- β receptors also cross-talk with oncogenic signaling pathways. For example, TGF-β can activate the Ras-MAPK pathway, which further increases TGF-β expression and cell growth. In addition, snail1 induced by the PIK3CA-Akt-mTOR pathway can be used as an effector protein TGF-β-related EMT. TGFβ pathway has dual anti- and pro-tumoral roles at the cancer cell level, depending on tumor stage and genetic alteration background, with mechanistic differences between cancer models. The current paradigm for the role of TGFβ in carcinogenesis at the pancreatic cancer cell level is that accumulation of genetic alterations in the TGFβ pathway drives the pathway's evolution from tumor-suppressive to tumor-promoting activities.

1.4 Wnt signaling pathway

WNT signaling is required for regulation of growth, differentiation, and cell death in normal epithelial cells. However, hyperactivated WNT signaling has also been implicated in proliferation, survival, and ability to metastasize in pancreatic cancer. Up to 65% of patients with pancreatic cancer are detected abnormally activated Wnt pathways. Since the severity of IPMN dysplasia is associated with up-regulated Wnt signaling, its abnormality may be a later event in the progression of pancreatic cancer. At the same time, Wnt pathway is more active in pancreatic cancer stem cells, suggesting that Wnt signaling plays an accelerating-multi-phase role in the development of exocrine pancreatic cancer. When Wnt signaling is activated, CK1ε and DVL (Dishevelled Segment Polarity protein) are able to dissolve the β-catenin degradation complex, which results in removal of inhibition signal on β-catenin. An inhibitory complex composed of glycogen storage kinase 3 (GSK3) can inactivate β-catenin in normal cellular homeostasis. Moreover, through the ubiquitin-protesome pathway, adenomatous polyposis coli (APC) and Axin proteins are capable of degrading β-catenin. Thus, if upstream Wnt signaling inhibits the degradation process, β-catenin is stabilized in the cytoplasm and then moves to the nucleus to initiate transcription of certain genes such as c-myc, cyclin D1 and MMP-7, which in turn leads to cancer cell infiltration, metastasis and drug resistance.

1.5 Sonic hedgehog signaling (SHH) pathway

There are two signal receptors in the SHH signaling pathway: patched and smoothened. In the absence of SHH, a polypeptide ligand in the cell, activation of patched receptors inhibits smoothened receptors, which is a major signal transduction pathway. However, once the ligand binds to the smoothened receptor, the smoothened receptor is activated and releases a transcription factor called Gli1. Gli1 is capable of stably transferring to the nucleus and inducing transcription of target genes such as Wnt, JAG2, Snail and stem cell markers, CD44 and CD133, as well as other genes involved in cell growth and EMT processes. Similar to Wnt signaling, SHH pathway also plays a role in different stages of pancreatic cancer development. SHH signaling is involved in the induction of malignant potential in pancreatic cancer, controlling processes of proliferation, invasiveness and tumorigenesis. At the same time, it is also capable of maintaining pancreatic cancer stem cells, and may be activated under conditions of tumor hypoxia. Thus, SHH signaling pathway may represent a potential therapeutic target for patients with refractory pancreatic cancer and the use of its inhibitors will likely play an important role in future therapeutic strategies.

1.6 Notch signaling pathway

The Notch signaling pathway has been known to play critical mechanistic roles in the development of organs, tissue proliferation, differentiation and apoptosis. Current studies suggest that Notch mediates EMT processes and promotes cancer cell metastasis to distant organs in the presence of TGF-α stimulation in the pancreas. Without Notch signal, DNA binding protein CSL (RBPJκ in mammalian cell, recombination signal binding protein for immunoglobulin κ J region) acts as a transcriptional repressor to inhibit the transcription of Notch signaling pathway target gene. When Notch signal is present, the Notch receptor changes its conformation following binding to the ligand, occurs proteolysis mediated by γ-secretase, and releases intracellular domain of Notch (NICN) with nuclear localization signal. NICN is transferred to the nucleus to bind to the transcriptional regulator CSL, thereby activating the corresponding target genes such as hes, hey, and the like. Other studies show that Notch signaling pathway has a CSL-independent pathway in addition to a CSL-dependent pathway. Overall, Notch signaling plays an important role in tumor progression. Aberrant Notch signaling leads to pancreatic cancer although with different net effects (oncogenic vs. oncosuppressive), depending on tumor subtypes, the timing, intensity and interaction with other signaling pathways. Notch signaling pathway regulates pancreatic cancer stem cells and is involved in pancreatic CSCs self-renewal. Therefore, through the regulation of the Notch signaling pathway, it would likely be successful in the elimination of CSCs and be useful for the eradication of pancreatic tumor recurrence and metastasis.

2 Pancreatic cancer diagnosis

Contrary to the general belief that pancreatic ductal adenocarcinoma (PDAC) is a rapidly growing malignant tumor, its average time from precancerous lesions to metastatic cancer may take up to 20 years. It is a very important result and suggests that there may be sufficient time to screen for the key target lesions earliest stage cancer and those precancers at greatest risk of progression. Over the past decade, genomics, epigenetics, and proteomics have great advancement, which accelerates the identification of candidate biomarkers to enable the detection of PDAC at an early stage.

2.1 Molecular Markers for Pancreatic cancer

The mutated genome of PDAC is complex. In recent years, in-depth analysis on genome coding region by high-throughput studies of gene expression revealed a large number of abnormal expression of genes associated with PDAC. Common mutant genes for PDAC include KRAS, TP53, SMAD4 and CDKN2A. KRAS has a mutation rate of 75%-95% in PDAC, which makes it the most common mutation, but it is relatively insensitive to early PDAC and has other interfering factors. At the same time, low circulating concentration of tumor DNA in the blood at early cancer stage is another challenge for this early diagnosis. Currently, genes that have been identified for abnormal methylation during pancreatic cancer include p16, ppENK, cyclin D2, SPARC/osteonectin SOCS-1, TSLC1, and the enrichment of some abnormal methylation genes in the genome of pancreatic cancer patients seems to be a single high-level discriminant marker of PDAC. Meanwhile, plentiful mi-RNAs are considered to be potential biomarkers of pancreatic cancer, and PDAC-specific microRNA profiles have been found in serum, pancreatic tissue, cyst fluid, and whole blood. At the same time, abnormal methylation genes of patients with PDAC include ADCY1, CD1D, BMP3, of which ADCY1 is the most sensitive monomethylation marker.

Pancreatic intraepithelial neoplasia (PanIN) is considered to be ductal lesions that are precursors of invasive cancer. Overexpression of HER-2/neu and point mutations of KRAS gene are early events that can be used to distinguish between low-grade ductal precancerous lesions (PanIN-1) and normal ductal epithelial cells. Inactivation of the p16 gene is more common in high-grade PanINs and seems to occur frequently in the mid-stage of tumorigenesis, while inactivation of p53, DPC4 and BRCA2 is a relatively later event in the progression of tumor. With regard to these detection markers, further validation is required in subsequent research to develop their molecular diagnostic function in the clinic as soon as possible.

2.2 Protein Markers for Pancreatic cancer

Carbohydrate antigen 19-9 (CA19-9) is the most widely studied routine biomarker for PDAC, which is usually normal in early disease. CA19-9 has sensitivity and specificity only within a certain range, with an average sensitivity of 80% and a specificity of 86%. However, CA19-9 also has its defects. In some non-malignant tumor diseases, such as acute cholangitis and pancreatitis, it may be accompanied by an increase in CA19-9. At the same time, other malignant tumors may also be associated with elevated CA19-9, such as 2/3 of patients with cholangiocarcinoma and 1/2 of patients with hepatocellular carcinoma. Compared with moderately and highly differentiated pancreatic cancer, the poorly differentiated pancreatic cancer has no obvious increase in CA19-9. Moreover, CA19-9 has insensitivity to early pancreatic tumors and precancerous lesions. Therefore, CA19-9 is not currently used as a biomarker for screening asymptomatic populations, but is used for examinations in patients with pancreatic cancer, to help doctors identify patients with pancreatitis or pancreatic cancer and detect tumor recurrence after surgical resection.

Carcinoembryonic antigen (CEA), the first tumor marker used to detect pancreatic cancer, is a glycoprotein. One report shows an average sensitivity of 54% and an average specificity of 79% for CEA. Also, in some other tumors, such as breast cancer, stomach cancer, and colorectal cancer, there is also CEA expression. Therefore, low sensitivity and poor specificity make CEA not be used for screening for pancreatic cancer. In the past 20 years, CEA has been replaced by CA19-9 in the diagnosis of pancreatic cancer. At the same time, considering that CEA cannot be used independently in the diagnosis, its clinical application may require the combination of other markers. Other blood circulating proteins that have been identified as candidate biomarkers include OPG, ICAM-1, TIMP-1, MIC-1, S100 P, and the like.

Table 1 Molecular and protein detection markers in pancreatic cancer

3 Targeted Therapy for Pancreatic Cancer

Because the molecular pathogenesis of pancreatic cancer is very complex, its occurrence, development and metastasis are closely related to the abnormality of various gene mutations and cell signaling pathways. These molecular mechanisms provide a number of potential key targets for the treatment of pancreatic cancer. Therefore, a single molecular targeted drug or combined with chemotherapy drugs has been a research hotspot. It includes mainly therapies targeting against kinases, including epidermal growth factor receptor, Ras/Raf/mitogen-activated protein kinase cascade, human epidermal growth factor receptor 2, insulin growth factor-1 receptor, phosphoinositide 3-kinase/Akt/mTOR and hepatocyte growth factor receptor. So far, gemcitabine is considered the standard chemotherapy for advanced pancreatic cancer. In Table 2-12, selected clinical trials of novel therapeutic targets for the treatment of pancreatic cancer are presented.

3.1 Pancreatic cancer therapy for RTK pathway

Therapies involving anti-EGFR monoclonal antibodies include cetuximab, a chimeric IgG1-type, and panitumumab, a humanized IgG2-type antibody. These antibodies reversibly inhibit the tyrosine kinase domain of EGFR by competitive binding of ATP. Erlotinib is a small inhibitor of EGFR, and vatalanib is an oral poly-tyrosine kinase inhibitor with strong affinity for platelet-derived growth factor and vascular endothelial growth factor (VEGF) receptors (VEGFRs). Trastuzumab, a humanized direct antibody against HER2 kinase which is based on Akt phosphorylation decrease and EGFR/HER2 heterodimerization. Trametinib is a drug that works by binding to and blocking certain proteins MEK1 and MEK2 from working. Crizotinib is an inhibitor of c-Met that has a role in reducing tumor progression and metastasis, and cabozantinib is a potent dual inhibitor of c-Met and VEGFR-2 signaling and tumor stem cell markers. AMG102, a monoclonal antibody against human HGF. Everolimus is mTOR inhibitors and in clinical trials. Rigosertib is a small molecular inhibitor of PI3. Bevacizumab, a recombinant humanized monoclonal anti-VEGF antibody, prevents VEGF binding to VEGFR-1 and VEGFR-2. Vatalanib (PTK787) and Axitinib (AG-013736) are small molecule inhibitor aginst VEGF and show to be very active in preclinical studies.

Table 2 Clinical trials of EGFR mAb Cetuximab

Table 3 Clinical trials of EGFR mAb Panitumumab

Table 4 Clinical trials of EGFR inhibitor Erlotinib

According to statistics, a total of 10 Erlotinib projects targeting pancreatic cancer EGFR are currently in clinical stage, of which 3 are recruiting and 7 are not recruiting.

Table 5 Clinical trials of HER2 mAb Trastuzumab

Table 6 Clinical trials of MEK inhibitor Trametinib

Table 7 Clinical trials of c-Met inhibitor Crizotinib

Table 8 Clinical trials of c-Met&VEGFR-2 inhibitor Cabozantinib

Table 9 Clinical trials of mTOR inhibitor Everolimus

According to statistics, a total of 18 Everolimus projects targeting pancreatic cancer EGFR are currently in clinical stage, of which 10 are recruiting, 7 are not recruiting and 1 is Enrolling by invitation.

Table 10 Clinical trials of VEGF mAb Bevacizumab

According to statistics, a total of 18 Bevacizumab projects targeting pancreatic cancer EGFR are currently in clinical stage, of which 6 are recruiting and 12 are not recruiting.

3.2 Pancreatic cancer therapy for JNK pathway

SP600125 is a reversible ATP-competitive inhibitor. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-γ, TNF-α, and prevented the activation and differentiation of primary human CD4 cell cultures.

3.3 Pancreatic cancer therapy for TGF-β/SMAD pathway

AP 12009 (trabedersen) is a phosphorothioate antisense oligodeoxynucleotide specific for the mRNA of human Transforming Growth Factor beta 2 (TGF-beta-2). SD-208, an inhibitor of TGFβRI kinase, reduced pancreatic cancer growth and metastasis in vivo and reduced fibrosis in the tumor microenvironment. LY2109761, a dual inhibitor of TGFβRI/II kinase and galunisertib (LY2157299), an inhibitor of TGFβRI kinase.

Table 11 Clinical trials of TGFβRI inhibitor galunisertib

3.4 Pancreatic cancer therapy for Wnt pathway

OMP-54F28(vantictumab) is a recombinant fusion protein combining human FZD8 receptor with the ligand binding domain and human IgG1 Fc fragment, which promotes a marked differentiation of tumor cells that is coupled with profound reduction in tumorigenic potential. OMP-18R5, is a monoclonal antibody that interacts with 5 frizzled receptors, thus blocking the induction of WNT cascade by its ligand.CGP-04909, ICG-001 and PRI-724 can inhibit β-catenin induced transcription and CREB binding. G007-LK, G244-LM, IWR-1, JW55 and XAV939 can inhibit tankyrase leading to Axin stabilization.

3.5 Pancreatic cancer therapy for SHH pathway

Saridegib(IPI-926) is a inhibitor of Sonic hedgehog which act on SMO, promotes the expression of several target genes responsible for desmoplastic reactions and releases inhibition of pancreatic cell autophagy. GDC-0449 and BMS-663513 are also orally bio-available selective inhibitors of SMO. LDE225 erismodegib), a SMO antagonist, can suppresses tumor growth. GANT-61, a Gli transcription factor inhibitor, has been shown to inhibit pancreatic cancer stem cell growth.

Table 12 Clinical trials of SMO inhibitor GDC-0449

Table 13 Clinical trials of SMO antagonist LDE225

3.6 Pancreatic cancer therapy for Notch pathway

RO492909 and MK-0752 are γ-secretase inhibitors that inhibit hydrolysis of Notch protein.