**5. Pancreatic cancer treatment**

#### **5.1. Chemotherapy**

PCSC markers (c-Met, ABCB1, and CD44), which decrease their expression. Oncogenic miR21

Leptin can directly regulate the expression of HDAC4 and HDAC5 and indirectly affect the expression of other HDAC via microRNA or PCSC markers. We have suggested that leptin can increase the expression of miR21, which in turn can increase the expression of HDAC3. Analysis of data from PC biopsies (TCGA databank) suggested that HDAC, miRNA21/200, and leptin could have complex signaling crosstalk that could be a novel therapeutic target for obese PC patients. We further determined the effects of leptin on HDAC expression in PC tumorspheres. HDAC3 and HDAC8 expression was increased by leptin. Furthermore, the Gemcitabine-induced decreased expression of HDAC2, HDAC3, and HDAC8 was reversed by leptin. Thus, we have shown that leptin through its effects on PCSC, ABCB1, and HDAC could be involved in PC chemoresistance [65]. Moreover, using another chemotherapeutic agent commonly used in PC treatment, 5-FU, we demonstrated that leptin impaired 5-FU cytotoxicity by increasing the expression and number of PCSC+, pluripotency+, and EMT+ PC cells. ABCC5 and ABCC11 expression as well as the number of positive cells for these ATPbinding cassette proteins were increased by leptin in PC tumorspheres. These leptin's effects protected the survival of PC tumorspheres treated with 5-FU and reduced its cytotoxicity. The survival of PC tumorspheres treated with 5-FU and leptin was linked to reduced apoptosis. Leptin increased the levels of PARP, Bcl-XL, and RIP and decreased Bax. 5-FU increased caspase-3 activation, which was reduced by leptin. These data could help to unravel the multiple

mechanisms through which leptin signaling contributes to drug resistance in PC [30].

Notch signaling controls the cell proliferation, PCSC maintenance and differentiation, apoptosis, invasion, and metastasis in cancer. Overexpression of Notch receptors (Notch1 and Notch2) was found in PCSC when compared with nonmalignant pancreatic stem cells [66]. DLL4 increase in PC cells stimulated the expression of Oct-4, Nanog, and stem cells [67]. PCSCs that express Oct-4, Sox-2, and Nanog show an increased aggressivity and chemoresistance. Notch4 overexpression was linked to PC chemoresistance to Docetaxel [68]. Expression of Notch3 and Hey1 was associated with reduced survival in PC [69]. Resistance to Gemcitabine correlated with Notch2, Notch4, and JAG1 overexpression [70]. The inhibition of Notch1 by siRNA suppressed proliferation, induced apoptosis, and reduced migration and invasion of PC cells [71]. Notch signaling induced EMT phenotype in Gemcitabine-resistant PC cells overexpressing Notch2, Notch4, and JAG1. Furthermore, the inhibition of Notch signaling decreased EMT markers, including Vimentin, Snail, Slug, and ZEB1, in human PC cell lines [72]. MiR200 members increased Notch activation by ZEB1 that regulates the expression of JAG1 and the mastermind-like coactivators (Maml2 and Maml3). In PC cells, miR200 expression showed an inverse correlation with JAG1 and ZEB1 levels [73]. Therefore, miR200 inhibits EMT by interacting with ZEB1/2 and the Notch pathway and represses self-renewal and differentiation in

Our data showed that leptin induced the expression of Notch family components in PC (Notch1–4, DLL4, JAG1, survivin, and Hey2), PCSC markers (CD24CD44ESA, ALDH, CD133,

increases the expression of ABCB1, ALDH, and CD44.

40 Advances in Pancreatic Cancer

*4.7.1. Leptin-Notch crosstalk in pancreatic cancer*

CSC. MiR200 is also involved in apoptosis [72].

To decrease the risk of local and distant metastasis, adjuvant therapy is usually started 1–2months after PC surgery. Although no regimen has been proven significantly more effective than others, a regimen based on 5-FU or Gemcitabine for 6 months is usually the option used to reduce PC patients' mortality [75]. The activity of 5-FU/Leucovorin has been compared to Gemcitabine as an adjuvant therapy in the European Study Group for PC (ESPAC)-3 trial [76]. However, the study showed that median overall survival for patients treated with 5-FU/ Leucovorin was 23 months when compared with 23.6 months for patients treated with Gemcitabine. The ESPAC-4 study measured the efficacy of a combination treatment with Gemcitabine plus Capecitabine when compared with monotherapy with Gemcitabine alone. The results showed a survival of 28 months in the combined therapy when compared with 25.5 months in the monotherapy group. Because the dual therapy was well tolerated, the combination of Gemcitabine and Capecitabine has been used as a standard in the clinical setting [77]. Currently, regimens with Gemcitabine plus nanoparticle albumin-bound Paclitaxel (nab-Paclitaxel) and a combination of 5-FU, Irinotecan, and Oxaliplatin (FOLFIRINOX) are evaluated in the clinical setting [78]. Gemcitabine has usually some efficacy as an adjuvant therapy, but often patients develop chemoresistance. Nab-Paclitaxel, a water-soluble compound, has enhanced distribution properties within the tumor microenvironment when compared with Paclitaxel. However, studies have shown that nab-Paclitaxel treatment neither decreased tumor stroma nor increased tumor vascular perfusion in a mouse patient-derived xenograft (PDX) tumor model [79]. The infiltration of neoplastic lesions by CD8+ T lymphocytes is associated with improved prognosis. However, a CD40 monoclonal antibody that activated CD8+ T cells in Phase I clinical trial had only a partial response [80]. FOLFIRINOX and nab-Paclitaxel plus Gemcitabine have the potential to downstage local advanced disease and to improve tumor resection rates. The use of chemoradiation therapy as an adjuvant is controversial and with minimal effects on survival in clinical trials so far [81]. New studies that incorporate modern radiation techniques and current chemotherapy regimens are still needed to determine if radiation is beneficial in PC treatment.

### **5.2. Targeted therapy**

A comprehensive genetic analysis of PC showed that these tumors contain an average of 63 genetic alterations in 12 cellular signaling pathways, including Notch pathway [82]. A Phase Ib trial for PC using a combination of Demcizumab (OMP-21M18), a monoclonal antibody against Notch ligand, DLL4, with Gemcitabine and Abraxane, showed some clinical benefits [60]. An antibody against Notch2 and Notch3, Tarextumab, was tested in Phase 2 clinical trials in combination with Gemcitabine and nab-Paclitaxel in patients with metastatic PC. For these patients, the median progression-free and overall survival were 5.6 and 11.6 months, respectively. Gamma secretase inhibitors (GSIs) have been used in clinical trials in PC. For example, a GSI called RO4929097 was safely tolerated in combination with Gemcitabine and achieved clinical antitumor activity and more than 4 months of stable disease. However, the use of GSI has limitations and still represents a challenge because of the increased drug toxicity and lack of high specificity to Notch besides other substrates of γ-secretase [83].

Desmoplasia is a target in PC treatment. Hyaluronan, a component of the ECM of PC, is a naturally occurring nonsulfated glycosaminoglycan that was targeted using pegylated hyaluronidase (PEGPH20). In a Phase II study combining Gemcitabine, nab-Paclitaxel, and PEGPH20, there was no difference seen in the survival of PC patients that had this addition to their treatment. Also, due to the ubiquitous nature of hyaluronan, there were unexpected side effects, such as thrombosis. For the Gemcitabine, nab-Paclitaxel, and PEGPH20 study, a subset analysis was performed on the high-hyaluronan patients. In the arm receiving PEGPH20, the response rate was 45% when compared with 31% in controls, which was encouraging, and led to a Phase III clinical trial (HALO301) for patients that had high hyaluronan. In these studies, Lovenox was included for anticoagulation [84].

STAT3 inhibition has been shown to decreased PC growth in mouse models. Napabucasin decreased STAT3 transcription and tumorsphere formation and showed some efficacy in PC. Napabucasin induced a median progression-free survival of >7.1 months and a median overall survival of >10.4 months in PC patients. Based on these encouraging results, it is now being evaluated in a PC Phase III study in combination with Gemcitabine and nab-Paclitaxel (NCT02993731) [85].

with 5-FU and leptin, IONP-LPrA2 reduced tumorsphere formation and cell proliferation, the number of Notch+, ABCC5/11+, and PCSC+ cells, and increased apoptosis. Thus, IONP-LPrA2 resensitized PC cells to 5-FU actions [28, 30]. In view of leptin multiple effects on PC and the involvement of Notch signaling in leptin's effects, targeting leptin-Notch crosstalk in PC patients might be a new treatment strategy for this deadly disease (**Table 1**). The addition of leptin antagonists to current chemotherapeutic treatment could represent a new strategy to

Pancreatic Cancer, Leptin, and Chemoresistance: Current Challenges

http://dx.doi.org/10.5772/intechopen.76400

43

**Table 1.** Inhibition of leptin signaling using IONP-LPrA2 resensitizes PC cell lines to chemotherapy.

PC is a lethal systemic disease that is difficult to detect and treat. This is mainly due to the fact that even patients diagnosed with early stages eventually develop metastasis. The deep abdominal position of the pancreas is an additional factor that delays the onset of specific PC symptoms. Early PC diagnosis and potential cure remain important challenges due to the lack in screening methods and specific biomarkers. PC risk factors, such as high-fat diet, obesity, tobacco, and alcohol consumption, can be modified, leading to prevention of disease occurrence and

overcome drug resistance and to improve survival of PC patients.

**6. Conclusions**

The expression of leptin in gastroesophageal adenocarcinomas was associated with chemoresistance. Therefore, the addition of leptin antagonists to current chemotherapeutic treatment could represent a new strategy to overcome drug resistance and to improve survival of PC patients. SHLA, a leptin antagonist, increased the sensitivity of resistant gastric cancer cell line, AGS Cis5, and the esophageal adenocarcinoma, OE33, to cisplatin [86].

LPrA2 was designed and tested in vitro and in vivo in PC xenograft mouse models in our laboratory. LPrA2 is composed by a leptin sequence corresponding to its binding Site III of the leptin molecule. LPrA2 was conjugated to iron-oxide nanoparticles (IONP-LPrA2) to increase its bioavailability and effectiveness to block leptin signaling in cancer cells [28]. IONP-LPrA2 showed no toxicity and did not affect energy balance (body weight or food intake) or general health when it was administered to mice. IONP-LPrA2 reduced the expression of Ob-R, Notch, and PCSC markers. Furthermore, specific inhibition of leptin signaling by IONP-LPrA2 delayed tumor onset and decreased tumor growth in a PC xenograft mouse model. Our data also showed that IONP-LPrA2 could be used as an adjuvant therapy to 5-FU. In PC cells treated


**Table 1.** Inhibition of leptin signaling using IONP-LPrA2 resensitizes PC cell lines to chemotherapy.

with 5-FU and leptin, IONP-LPrA2 reduced tumorsphere formation and cell proliferation, the number of Notch+, ABCC5/11+, and PCSC+ cells, and increased apoptosis. Thus, IONP-LPrA2 resensitized PC cells to 5-FU actions [28, 30]. In view of leptin multiple effects on PC and the involvement of Notch signaling in leptin's effects, targeting leptin-Notch crosstalk in PC patients might be a new treatment strategy for this deadly disease (**Table 1**). The addition of leptin antagonists to current chemotherapeutic treatment could represent a new strategy to overcome drug resistance and to improve survival of PC patients.
