Therapeutic Approaches in Pancreatic Cancer: Recent Updates

Cancer is a significant challenge for effective treatment due to its complex mechanism, different progressing stages, and lack of adequate procedures for screening and identification. Pancreatic cancer is typically identified in its advanced progression phase with a low survival of ~5 years. Among cancers, pancreatic cancer is also considered a high mortality-causing casualty over other accidental or disease-based mortality, and it is ranked seventh among all mortality-associated cancers globally. Henceforth, developing diagnostic procedures for its early detection, understanding pancreatic cancer-linked mechanisms, and various therapeutic strategies are crucial. This review describes the recent development in pancreatic cancer progression, mechanisms, and therapeutic approaches, including molecular techniques and biomedicines for effectively treating cancer.


Introduction
Pancreatic cancer is a fatal disease that affects the pancreas, a large gland in the abdomen. American Cancer Society suggested that pancreatic cancer is the third highly common type of cancer in humans. Initial recognition is critical so that patients receive the extremely efficient treatment possible and have the greatest chance of survival [1][2][3]. Primarily, many patients are detected at an advanced stage. In the United States, this year approximately 53,090 people will be diagnosed with pancreatic cancer, and more than 41,170 people died from the disease [4,5]. Therefore, we must continue identifying new and improved ways to diagnose the disease earlier to save lives. Pancreatic cancer is highly destructive and has a poor prognosis with only a 9% of survival rate within five-years. Unluckily, the symptoms of pancreatic cancer are often vague and easy to overlook, so many people do not know that they have it until it is too late [6,7]. Its primary symptoms include jaundice, loss of appetite/weight, and right abdomen pain [4,8]. The pancreas is also challenging to reach and diagnose, so traditional imaging methods often cannot detect cancer early enough. However, early diagnosis and treatment are critical to improving survival. Today's early detection methods are better than ever, but there are still significant limitations to available diagnostic tools. Methods of detecting pancreatic tumors include x-rays, ultrasounds, MRI.s, and CT scans [4]. These tests can effectively identify the tumor's location and the extent of the disease. It is difficult to diagnose early, but specific blood tests can detect it if certain risk factors exist. Specific biomarkers can also be applied to detect cancer cells [9,10]. Pancreatic cancer is diagnosed with endoscopy, a minimally invasive procedure that uses a tube with a tiny camera attached to examine the inner lining of the digestive tract. During the process, removing a tissue sample from the pancreas to test for cancer cells can be validated [11,12]. Further, staging determines how far your cancer has

Pancreatic Cancer Molecular Manifestation and Pathways Regulation
Pancreatic cancer is challenging, with a poor progression and limited treatment options [38]. Its tumorigenesis or metastasis involves, pathways, including phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), RAS, janus kinase (JAK)/signal transducer, and activator of transcription (STAT), NF-κB, Hippo/yes-kinase-associated protein (hippo/YAP), and Wingless/int1 (WNT). These pathways are associated with numerous cellular processes linked to pancreatic cancer, such as apoptosis, angiogenesis, differentiation, immunological regulations, metabolism, migration, and cell proliferation. In addition, histone modification is a vital feature in pancreatic cancer for epithelial-to-mesenchyme transition [4,39]. The regulation and consideration of these pathways for pancreatic cancer can be helpful in developing novel targets and therapeutics.
A key feature of pancreatic cancer includes the immunosuppressive tumor microenvironment [40,41]. Several molecular and cellular factors have been identified as critical players in the induction and maintenance of immunosuppression within the pancreatic stroma. The pancreatic stroma is composed of an extracellular matrix, immune cells, and fibroblasts that surround the tumor cells, forming a barrier that impedes cancer drug effect and immune cell infiltration [42] (Figure 1). Macrophages, which are a critical type of innate immune cells, play a significant role in immunomodulation in pancreatic cancer via a secreting range of cytokines [43]. Cytokines play a crucial role in tumor growth and immune cell evasion by promoting cancer cell invasion, proliferation, and immunosuppression [44,45]. Inflammatory cytokines such as IL-1β, IL-6, IL-8, and IL-10, have been demonstrated to activate tumor-associated macrophages (TAMs). The cytokines and chemokines attract immune cells, i.e., regulatory T cells (Treg cells), TAMs, and neutrophils, which impede CD8+ cytotoxic T cells function. Regulatory T cells (Tregs) have been associated with advancing pancreatic cancer by curbing cytotoxic T cells [46]. Pancreatic cancer cells coordinate immune evasion by synchronizing the secretion of cytokines in a highly coordinated way through TP53-dependent or KRAS-dependent pathways [47]. Tregs are vital in preserving immunologic self-tolerance and regulating suppression in pancreatic tumor growth. and immune cell infiltration [42] (Figure 1). Macrophages, which are a critical type of innate immune cells, play a significant role in immunomodulation in pancreatic cancer via a secreting range of cytokines [43]. Cytokines play a crucial role in tumor growth and immune cell evasion by promoting cancer cell invasion, proliferation, and immunosuppression [44,45]. Inflammatory cytokines such as IL-1β, IL-6, IL-8, and IL-10, have been demonstrated to activate tumor-associated macrophages (TAMs). The cytokines and chemokines attract immune cells, i.e., regulatory T cells (Treg cells), TAMs, and neutrophils, which impede CD8+ cytotoxic T cells function. Regulatory T cells (Tregs) have been associated with advancing pancreatic cancer by curbing cytotoxic T cells [46]. Pancreatic cancer cells coordinate immune evasion by synchronizing the secretion of cytokines in a highly coordinated way through TP53-dependent or KRAS-dependent pathways [47]. Tregs are vital in preserving immunologic self-tolerance and regulating suppression in pancreatic tumor growth. Recent studies have emphasized the vital importance of the cancer cell microenvironment in advancing cancer progression [48]. Moreover, the immune cells within the cancer cell microenvironment, including B cells, natural killer cells, T cells, and myeloid- Recent studies have emphasized the vital importance of the cancer cell microenvironment in advancing cancer progression [48]. Moreover, the immune cells within the cancer cell microenvironment, including B cells, natural killer cells, T cells, and myeloid-derived suppressor cells are dysregulated and fail to mount an effective antitumor response [49]. The immunosuppressive cancer micro-environment shows a decisive role in the advancement of therapeutic resistance, which explains why current treatments are ineffective against pancreatic cancer. The progression of pancreatic cancer is modulated by various signaling pathways, including NF-κB, JAK/STAT, PI3K/AKT, Hippo/YAP, RAS, and WNT pathways (Figure 2). These pathways impact cellular functions such as apoptosis, differentiation, immunological regulation, metabolism, migration, angiogenesis, and cell proliferation [50][51][52][53]. Exploring and managing these signaling pathways has the potential to uncover novel targets and therapies for pancreatic cancer. RAS is a crucial driver of effector pathways, and its oncogenic activation is frequently found in pancreatic cancer, particularly in the KRAS isoform [47]. This activation can lead to cell proliferation, transformation, metastasis, and pro-inflammatory signaling activation. The PI3K/AKT pathway plays a significant role in pancreatic cancer regulation, with great potential for therapeutic targeting [54]. The PI3K/AKT pathway is commonly triggered in pancreatic cancer, with abnormal AKT overexpression associated with poor prognosis. PI3K signaling controls pancreatic cell plasticity and is activated early in tumor evolution [55]. This pathway is also activated by insulin-like growth factors and abnormal expression of various noncoding RNAs [56]. Several PI3K/AKT inhibitors are being investigated for their potential therapeutic effects in pancreatic cancer patients. ineffective against pancreatic cancer. The progression of pancreatic cancer is modulated by various signaling pathways, including NF-κB, JAK/STAT, PI3K/AKT, Hippo/YAP, RAS, and WNT pathways ( Figure 2). These pathways impact cellular functions such as apoptosis, differentiation, immunological regulation, metabolism, migration, angiogenesis, and cell proliferation [50][51][52][53]. Exploring and managing these signaling pathways has the potential to uncover novel targets and therapies for pancreatic cancer. RAS is a crucial driver of effector pathways, and its oncogenic activation is frequently found in pancreatic cancer, particularly in the KRAS isoform [47]. This activation can lead to cell proliferation, transformation, metastasis, and pro-inflammatory signaling activation. The PI3K/AKT pathway plays a significant role in pancreatic cancer regulation, with great potential for therapeutic targeting [54]. The PI3K/AKT pathway is commonly triggered in pancreatic cancer, with abnormal AKT overexpression associated with poor prognosis. PI3K signaling controls pancreatic cell plasticity and is activated early in tumor evolution [55]. This pathway is also activated by insulin-like growth factors and abnormal expression of various noncoding RNAs [56]. Several PI3K/AKT inhibitors are being investigated for their potential therapeutic effects in pancreatic cancer patients. NF-κB is a crucial transcription-factor that shows a significant role in inflammation, is frequently stimulated in pancreatic cancer, and promotes cancer development, metastasis, and drug-resistance [57]. KRAS and other oncogenic mutations can also activate NF-κB. High quantities of chemokines are seen in pancreatic cancer, forming a positive feedback loop that enhances NF-κB signaling. Several ncRNAs can regulate the NF-κB pathway [58]. NF-κB is involved in antitumor immunity, and inhibitors of the pathway show promise as a therapeutic option. JAK/STAT pathways are engaged in a range of human cancers, together with pancreatic cancer, and increased JAK2 signaling indicates an inadequate prognosis of the disease [58,59]. The signaling pathway is implicated in inflamma- NF-κB is a crucial transcription-factor that shows a significant role in inflammation, is frequently stimulated in pancreatic cancer, and promotes cancer development, metastasis, and drug-resistance [57]. KRAS and other oncogenic mutations can also activate NF-κB. High quantities of chemokines are seen in pancreatic cancer, forming a positive feedback loop that enhances NF-κB signaling. Several ncRNAs can regulate the NF-κB pathway [58].
NF-κB is involved in antitumor immunity, and inhibitors of the pathway show promise as a therapeutic option. JAK/STAT pathways are engaged in a range of human cancers, together with pancreatic cancer, and increased JAK2 signaling indicates an inadequate prognosis of the disease [58,59]. The signaling pathway is implicated in inflammation in pancreatic cancer. In addition, interferons can increase PD-L1 expression via directly/indirectly influencing JAK-STAT signaling. Sustained JAK-STAT initiation can advance chronic inflammation and may impede CTL activation. JAK-STAT pathways have been associated with cancer developmental progress. YAP-TAZ are the major contributing factors of the Hippo pathway in pancreatic cancer [50]. Studies show that YAP is extremely articulated in patients and linked with an inadequate diagnosis of the disease. YAP is needed for cancer progression and is a crucial player in KRAS mutant mice. YAP can lead to disease relapse in the deficiency of KRAS and plays a vital driver of squamous pancreatic cancer. YAP-TAZ are transcriptional co-activators that can drive the gene expression engaged in proliferation and cell survival, impacting the hostile behavior of pancreatic cancerous cells. YAP also regulates the immunosuppressive microenvironment by modulating the behavior of PSCs and influencing the enlistment of TAMs and MDSCs. The WNT pathway controls somatic stem cells and is involved in pancreatic carcinogenesis and tumor progression through canonical and noncanonical pathways [60,61]. KRAS activation promotes pancreatic cancer cell movement and infiltration through the WNT pathway, and increased WNT/β-catenin signaling enhances the stem cell-like phenotypes. Both canonical and noncanonical WNT ligands have been linked to pancreatic cancer progression [60].

Cyclin-Dependent Kinases
Protein kinases work an essential role in protein function alteration by phosphorylation to enhance or decline biological activities, i.e., transcription and translation [4,24]. However, protein kinase activity regulation is key in therapeutics against various diseases, including autoimmune, neural, and cardiovascular. Cyclin-dependent kinases (CDKs) such as CDK1-CDK4 and CDK6 serine/threonine kinases are primarily involved in cell cycle regulation by the mechanism of phosphorylation, interaction between proteins, or ubiquitin-based proteolysis. CDKs activation requires its binding to the cyclin subunit. To initiate cells from G2 to the mitosis phase, CDK1 complexes with cyclins A and B. On the other hand, CDK2 links to cyclins A and E, which is essential in the G1-S transition and in the S phase [24]. Alteration in cell cycle regulation is an essential phase towards the transition of normal cells to cancerous cells primarily associated with the functional de-regulation of CDKs. CDKs have been considered potential targets in cancer therapeutics. A few CDKs inhibitions are known, such as abemaciclib, dinaciclib, Palbociclib, ribociclib, and RO-3306, and are helpful in the treatment of cancer at metastatic or advanced cancer stage [24]. 4-benzyl-1piperazinyl-phthalazine compounds (8 g, 10 d and 10 h) are exhibiting anti-proliferation activity towards MDA-PATC53 with 50% of viability inhibitory concentration (IC 50 ) of 0.51-0.88 µM and PL45 with IC 50 of 0.74-1.14 µM). These phthalazines highly showed inhibitory selectivity against CDK1 with IC 50 of 36.8-44.5 nM. In contrast, a minor inhibitory influence of these compounds was noted against other kinases, including CDK2, CDK5, IGF1R, AXL, BRAF, FGFR, JAK1, and PTK2B [24]. 1-Piperazinylphthalazines (16 k, and 21 d) impeded VEGFR-2 with IC 50 of 0.35-0.40 µM [63]. In mechanism, cell cycle controlling mechanisms regulates the growth of cells. Inhibiting cell proliferation can be helpful to cancerous cell therapy targets by cell cycle arresting at G2/M check-points [24,63]. For cancer-treating drugs, the cell cycle arrest mechanism initiation is anticipated via their influence on cell toxicity. Phosphorylation of E2F1 and CDC6 by CDK2/cyclin A involves S phase termination and pushes the S phase to the G2 phase transition. Furthermore, CDK1 activation by cyclin A leads to cells being put in the M phase. During mitosis, the CDK1/cyclin B complex maintains CDK1 activity, which is vital for the progression of the cell cycle. Therefore, the alteration in CDK1 expression assists in separating chromosomes, mitosis accomplishment, and cytokinesis during cellular replication [24,63].

Oncolytic Virus
PDAC escape the antitumor immune-system through inadequate angiogenesis, massive-stroma, and immune-suppressive cells infiltration [7]. Consequently, a unique therapeutic strategy to accelerate the immune response against antitumor is considered necessary to enhance the efficacy of immunotherapy against PDAC. Inhibitors of immune checkpoints, such as antibodies towards anti-programmed cell death 1 (PD-1), are proven effective in cancer treatment [4,7]. In PDAC, the deprived immune response of PD-1 obstruction therapy is refractory. Therefore, novel oncolytic virotherapy is a potential therapeutic method via immunogenic cell death [64]. Oncolytic adenovirus OBP-702-mediate over p53 expression that can stimulate immunogenic cell death via antitumor immune responses towards human PDAC by releasing damage-associated molecular patterns, i.e., extracellular delivery of high mobility-group box 1 protein, and adenosine-triphosphate. A subcutaneous PAN02 tumor (PDAC) model suggested that OBP-702 involves tumor permeation of T cells (CD8 + ), and PD-1 blockade antitumor efficacy [64].
Mutation in a rat sarcoma virus (RAS) is associated with nearly 30% of the entire human cancers and accounts for almost 85% of Kirsten RAS (KRAS) of whole RAS mutations [65]. Pancreatic cancers are linked to the KRAS gene's oncogenic mutations in approximately 90% of cases. KRAS is a component of epidermal growth factor receptors (EGFR) signaling that are vital molecules in cancer therapy. In a mechanism, KRAS triggers the downstream growth-associated signaling pathways and quickly enhances phosphatidylinositol 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK). Cetuximab is used as a therapeutic towards EGFR in case of metastatic colorectal cancer individual who does not respond to chemotherapy. The application of cetuximab is restricted to wild-type KRAS. However, numerous attempts have been initiated to create molecules that act against tumors by inhibiting the activity of mutant KRAS [65]. A decline in specific miRNA's intracellular contents can provoke genetic information transmission that leads to disease progression. For example, compared to normal cells, the MIR143 expression that p53 transcribes declined by nearly 80% in colorectal tumors and PDAC. The mechanism of miRNA therapy involves rebuilding tumor cells to normal by substituting down-regulated miRNA or their suppression [65]. Chemically altered MIR143 consider-ably inhibits tumor growth by affecting AKT, KRAS, extracellular signal-regulated kinases (ERK), and son of sevenless homolog 1 (SOS1). In addition, chemically modified MIR143 (MIR143#12) anticancer effects involve inhibition of KRAS networks in pancreatic cancer cells with/without KRAS mutation and exhibit high-level RNA nucleases resistance activity [4,65]. miR-143 #12 showed superior anticancer activity (IC 50 = 0.36 nM) to siR-AKT, siR-KRAS, or siR-ERK2 silencing (IC 50 > 20 nM) toward BxPC-3 and MIA PaCa-2 cells. In contrast, PANC-1 exhibits higher IC50 of 4.75 nM for MIR143 than up to 0.98 nM for siR-AKT, siR-KRAS, or siR-ERK2 at the incubation of 96 h [65]. Here, MIR143 acts by cell cycle arrest autophagy or apoptosis. Overall, the cell proliferation inhibition was observed quite similarly for siR-KRAS and MIR143 towards pancreatic cancer KRAS mutant cells. Further, the effectiveness of MIR143#12 towards PANC-1 can be potentially improved by its supplementation with siR-KRAS/cetuximab. However, MIR143#12 showed negligible adverse influence on normal cell growth; thus, developing a suitable drug delivery system for its delivery towards specific tumors is essential for side effects assessment [65].

Nanomedicine
Nanomedicine is a promising approach that can be potentially applied for treatment, including cancer treatment. Still, the clinical use of nanomedicines is limited by various factors-(i) low loading of drugs, (ii) tumor target specificity, (iii) potential cytotoxicity, and (iv) desirable mass production. Regarding industrial and technical attributes, the alternative strategy for developing carrier-free prodrug nanoparticles seems more promising. A conjugate of doxorubicin (DOX) and cathepsin B specific cleavable peptide (FRRG) formulated via self-assembly followed Pluronic F68 stabilization as F68-FDOX prodrug nanoparticles exhibiting high loading of >50% drug [66]. F68-FDOX proficiently gathers within tumors via enhanced permeability and retention influence with further release of DOX via cathepsin-B specific cleavage process exhibited significant antitumor activity for MDA-MB231, HT29, and KPC960. In vivo analysis suggested that F68-FDOX is highly safe as no toxicity was noted toward normal cells [66]. Dextran-coated maghemite nanoparticles (56 µg/mL) exposure reduced~50% of PANC-1 cell viability after 72 h incubation via alteration in expressions of heat shock proteins (HSPs) and p53 protein [67]. Lipid nanoparticles (LNP DTX-P ) on gold nanoparticles with docetaxel prodrug showed enhancement in the uptake of nanoparticles up to 2.8-folds by MIA PACA-2 cells than the controls [68]. In vitro assay, MIA PACA-2 cells proliferation IC 50 of 9.8 nM for LNP DTX noted against only docetaxel prodrug (44.4 nM). In mechanism, nanoparticles containing cancerous cells cannot divide and are trapped in the M phase. Hence, LNP DTX-P on gold nanoparticles can be potentially employed for radiotherapy-based pancreatic cancer treatments [68]. The combined approach of using magnetic nanoparticles and hyperthermia against BxPC3 pancreatic tumor cells proved beneficial over nanoparticles only [69]. Nanoparticles enhanced DNA double-strand breaks via radio sensitization effects and ROS production. Here, a combined treatment strategy exhibited significantly higher BxPC3 cell death after 6 h of incubation than irradiation or nanoparticle administration [69]. MIA-PaCa-2 treated by 4 µM of polyethyleneglycol-betulinic acid (PEG-BA) polymer-drug conjugate exhibits enhancement in expression of proapoptotic genes TNF and CASPASE 3 up to 23.7-, and 12,060-fold, respectively [70]. BA helped in anti-inflammatory and antioxidant activities in this conjugate, and the conjugate showed IC 50 of 15.6 µM compared to the antioxidant potential for BA-only of >100 µM. These conjugates induce apoptosis-mediated death of MIA-PaCa-2 by arresting the sub-G1phase of cell cycles [70].

Adjuvants, Immunological Targets and Peptide
In Asia, S-1 adjuvant-based chemotherapy is primarily adapted to treat PDAC and its dose intensity highly influences survival potential [71]. Pancreatic cancer patients with S-1 dose intensity of ≥62.5% proved beneficial in survival with a median period of 53.3 months compared to its lower dose with a survival period of 20.2 months. This finding suggests that S-1 adjuvant chemotherapy is an effective therapeutic against pancreatic cancer for prolonged postoperative survival by maintaining a dose intensity above 60% [71].
Immune check-points inhibitors like antibodies PD-1/PD-L1 and CTLA-4 are applied for immunotherapy against cancers [64]. However, PDAC is not as sensitive for immunotherapy because of cytotoxic T cell inferior infiltration and limiting mutation burden. Immunological therapy that is associated with molecular compounds acting towards toll-like receptors (TLRs) can be a robust approach for treating cancers [19]. Erlotinib, an immunomodulatory drug linked with gemcitabine, works by impeding an epidermal-growth factor receptors tyrosine kinase. Compounds like protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) are emerging potential candidates against pancreatic cancer involving the TLR4 signaling pathway [19]. P-MAPA and gemcitabine-coupled therapy efficiently reduced the progression of neoplastic lesions and histopathological improvement by up to 80% in pancreatic cancer over only 40% repair using P-MAP alone. P-MAPA and P-MAPA/gemcitabine therapies enhance TLR4 protein contents that improve interferon signaling pathways via abnormal cell proliferation suppression [19].
In PDAC, thrombospondin-1 (TSP-1) associated with cancer-associated fibroblasts (CAFs) induces downregulation of Smad4 expression and enhances malignant potential via activation transforming growth factor-β (TGF-β) signals [72]. Smad4 involves TGF-β superfamily cytokines signal transduction by target genes transcriptional activation and plays a vital role in pancreatic tumor suppressor. Deletion of the Smad4 gene (DPC4) activates faster cell invasiveness and metastatic capability. In vivo, TSP-1) acts as an essential activator of TGF-β. In PDAC cells, DPC4 knockdown triggered TSP-1 overexpression along with TGF-β signal initiation. Also, the overexpression of TSP-1 caused Smad4 expression downregulation and improved the proliferation of cells. LSKL peptide (TSP-1 inhibitor) treatment prevents TSP-1 binding to a latency-associated peptide (LAP) and TGF-β signal activation that results in anticancer impacts via attenuation of cell proliferation, migration, and chemoresistance in PDAC cells. However, LSKL peptides as TSP-1 inhibitors can potentially be therapeutics in PDAC patients [72].

Natural Bioactive and Organic Compounds
Natural-derived biomolecules are proven to be beneficial for broad biotechnological applications [73][74][75]. Curcumin has been shown to have a broad target and involve in the regulation of many cellular signaling pathways such as NF-κB/COX2, WNT/β-catenin, SHH, STAT3, NOTCH, TGF/Smad, and PI3K/AKT. These are participating in the development and progression of various cancers [74][75][76][77][78][79][80]. Curcumin induces apoptosis, inhibition of progression, and angiogenesis in pancreatic cancer. For curcumin, IC 50 of 8 µM, 20 µM, and 12 µg/mL against BxPC3, and Panc1 has been noted, respectively [74,80,81]. It has been reported that curcumin down-regulates NF-κB binding, and the activity of Ikappa B kinase, which leads to decreased cell proliferation and enhanced apoptosis in pancreatic cancer [78]. Another study further supported this, where the author reported curcumin treatment with a pancreatic cancer cell line, improving the upregulation of FOXO1 expression [79,80]. Additionally, Curcumin has been displayed to pancreatic cancer cells to sensitization to gemcitabine drug, which failed to treat pancreatic cancer cells due to drug resistance [76,81]. Hence, curcumin was considered potential therapeutics for inhibiting the growth of pancreatic cancer stem cells.
Diphyllin is recognized as promising natural therapeutics against pancreatic cancer [88]. Still, its application in pancreatic cancer is limited due to partial solubility, inadequate potency, and weak metabolic stability. Therefore, the derivatization diphyllin can be beneficial to overcome these limitations. The nitrogen-based derivatization of diphyllin results in superior metabolic stability and aqueous solubility compared to pure diphyllin. The amino-derivatized compound 15 showed IC 50 of 3 nM against CFPAC-1 cells with 69-fold higher effectiveness over diphyllin as control [88]. These amino derivative compounds dose-dependently stimulated cell cycle arrest (G0/G1 phase) via CDK4 and cyclinD1 down-regulation. In the xenograft model, a compound 15 dose of 10 mg/kg treatment showed high potent efficacy against transplanted PANC02 tumors via growth suppression with no noticeable safety anxiety [88]. Diosgenin, a derivative of Solanum, Dioscorea, and Costus spp., is demonstrated to stimulate apoptosis and cell cycle arrest in pancreatic cancer cells PANC-1 and Patu8988. Furthermore, diosgenin downregulates an expression of oncogenic protein EZH2 and its target vimentin and upregulates PTEN [89,90]. Therefore, it could be a promising therapeutic compound for PC treatment targeting EZH2. In addition, 75 µg/mL diosgenin for 72 h has been reported to suppress 70% growth of Patu8988 and Panc-1 cell lies [83,89,90].

Microbiome
The human microbiome is a diverse ecosystem of microorganisms that inhabit the human body. Association of microbes is linked by signal molecules' communication [112][113][114]. The microbiome is estimated to consist of over 100 trillion microorganisms, the most common being bacteria, viruses, and fungi [115]. Recent studies advocate that the gut microbiome's balance and heterogeneity may contribute to the advancement and development of diseases, including pancreatic cancer [116,117]. Therefore, this section confers how the microbiome impinges on pancreatic cancer progression.
Previous findings have suggested that the microbiome can influence many aspects of human health, including the progression and advancement of pancreatic cancer [113,118,119]. Studies have shown that pancreatic cancer had significant alternation in beneficial bacterial species, such as Lactobacillus, Ruminococcus and Bifidobacterium, compared to healthy individuals and may promote the development of cancer cells [120]. Recent studies have suggested that Bifidobacterium may have a protective effect against pancreatic cancer. Some studies have found that individuals with higher levels of Bifidobacterium in their gut microbiome have a lower risk of developing pancreatic cancer [121]. The proposed mechanism for this protective effect is that Bifidobacterium may help to maintain a healthy gut environment by promoting a balanced gut microbiome and reducing inflammation. This is important because chronic inflammation is a common risk aspect for advancing pancreatic cancer [122]. Bifidobacterium may also have direct anticancer effects by producing compounds that inhibit the growth of cancer cells. For example, some studies have found that Bifidobacterium can produce short-chain fatty acids (SCFAs), which have been shown to have anticancer properties [123]. Lactobacillus may also has a protective effect against pancreatic cancer [124]. These organisms produce lactic acid and hydrogen peroxide, which may possess anticancer properties [125]. Regarding the progression of pancreatic cancer, studies have shown that some bacteria may be engaged in the cancer spread and the production of substances that can make cancer cells worse. Gram-negative bacteria can produce substances called exotoxins that damage the pancreas and cause cancer cells to grow [126]. Fusobacterium nucleatum, a species of Fusobacteria, is present at higher levels in the pancreatic tumors of patients with pancreatic cancer than in healthy individuals [127]. The microbiome of tumors has been proposed to be linked to the advancement of cancer and the ability of chemotherapy to be effective [35].
Moreover, recent studies have shown that the microbiota may also be directly or indirectly responsible for developing drug resistance in pancreatic cancer [120]. The main factor is the ability of microorganisms to absorb and metabolize some anticancer drugs, rendering them ineffective [128]. While additional work is necessary to elucidate the exact mechanism involved, understanding the microbiome may also help to prevent, diagnose, and treat this deadly disease or human health [128][129][130]. Bacteria in the gut may also produce metabolites that promote the growth of cancer cells. Gut bacteria have been found to produce N-nitroso compounds [131], which are known to be carcinogenic [132]. In addition, the research provided evidence that Helicobacter pylori seropositivity and increased risk of pancreatic cancer [133]. Another hypothesis is that host-microbiome dysbiosis may also contribute to the development of pancreatic cancer [134,135]. Studies have shown that patients with pancreatic cancer often have a lower diversity of gut bacteria than healthy individuals [136]. The gut microbiome is a complex ecosystem, and its changes may have unintended consequences. Therefore, further research is necessary to recognize the role of the gut microbiome in understanding pancreatic cancer molecular pathways and how it can be modulated to prevent or treat the disease.
3.8. Clustered Regularly Interspaced Short Palindromic Repeats, and Associated Protein 9 (CRISPR/Cas9) Significant progress has been made in developing new therapeutic modalities, including targeted, immune, and CRISPR/Cas9-guided therapy [137]. CRISPR/Cas9 technology is more efficient, less costly, and easier to implement than transcription activator-like effec-tor nucleases (TALENs), mega-nucleases (MNs), and zinc-finger nucleases (ZFNs) [138]. It allows for the deletion, replacement, or addition of genomic sequences by inducing non-homologous end joining/homology-directed repair. CRISPR/Cas9 is routinely employed to silence genes in pancreatic cancer development. Using CRISPR/Cas9, Watanabe et al. [139], showed that KDM6A-lacking cells display hostile behavior in human PDAC cell lines. Reduced extracellular vesicle secretion and a less robust motile phenotype were examined by knockouts of ANXA1 in Mia PaCa2 [140][141][142]. Furthermore, the knocking down of GALNT3 using CRISPR/Cas9s in Capan1 cells resulted in fewer tumor spheres losing their self-renewal capacity and migrating less efficiently [141]. Furthermore, knocking down SphK1 in PAN02 cells using CRISPR led to higher proliferation and migration [143]. Moreover, CRISPR/Cas9 technology was utilized to knock down C1GALT1 in PDAC cells; these cells were more aggressive regarding proliferation, migration, tumorigenicity, metastasis, and the expression of Tn and sTn [144].
Loss-of-function (LoF) phenotypic screens using a small-guide RNA (sgRNA) library are a powerful method for discovering new protein roles under certain growth conditions [145]. Thousands of plasmids encoding sgRNAs targeting individual genes are used in one massive screening activity known as CRISPR pooled library screening. First, the sgRNAs must be transduced into enough cells using a lentivirus, or retrovirus. Ultimately, the transduced cells are cultivated in a controlled environment to achieve a desired phenotype. Cell's DNA is extracted, and NGS is obtained after selection. The next step is for a computational tool like model-based analysis of genome-wide CRISPR-Cas9 knockouts to assess the NGS data by mapping it to a library containing the gene-specific gRNAs [145]. After transducing PANC1-cas9 to sgRNA library Brunello, several researchers have employed CRISPR assembled screening to target cancer [20]. The cells were then chosen with gemcitabine (100 nM) for six-days. Comparing the NGS data of selected samples than controls, PSMA6 was a differentially expressed gene. The subsequent research showed that PSMA6 suppression caused apoptosis and decreased spheroid development. Cells transduced with a TKO gRNA library and tested simultaneously revealed the fitness genes necessary for proliferation [20]. Using the BAGEL algorithm, each gene log Bayer factor (BF) was determined [146]. CTNNB1, LRP5, PORCN, TCF7L2, WLS, and the Frizzled (FZD) receptors FZD5, WNT7B, and WNT10A, were found to be necessary for HPAFII cell proliferation. This research was further confirmed in PaTU8988S and AsPC-1, establishing a Wnt pathway as a critical modulator for proliferations of cancer cells. In addition, the screening outcomes showed that Wnt-catenin signaling is specifically required in RNF43mutant PDAC proliferations and/or survivals. Genome-wide CRISPR-Cas9 knockouts screens were also done in PATU8988T and PATU8902 cells to discover genes involved in proliferation/survival in MAPKi presence [147]. CRISPR/Cas9 gene-editing system has applications in both in vitro and in vivo settings. The ex-vivo therapy would involve isolating cells, modifying them in a lab, and reintroducing them to the patient. Injecting genetic materials directly into the body is possible with in vivo therapy [148]. In vivo, it was generated by injecting adenoviral-Cre or lentiviral-Cre vectors retrogradely into pancreatic ducts. This method can potentially pave the way for novel pancreatic cancer treatments [148]. Although multiple CRISPR/Cas9 clinical trials have been conducted, and the technology holds great promise for treating monogenic disorders, degenerative diseases, and HIV infection, there is still a long way to go before it is applied to treat pancreatic cancer patients. Many obstacles are yet to be pondered. For instance, which genes are the most significant ones to focus on? Furthermore, accuracy, efficiency, and safety improvements are necessary to fulfill the needs of clinical applications in pancreatic cancer.

Conclusions
Pancreatic cancer is primarily constrained by early diagnosis or potential therapy procedures. It is a potentially fatal disease that can spread to other body parts. If caught early, the disease can be treated with surgery and chemotherapy. However, some resist these treatments or do not respond to them at all. Fortunately, the cancer is responsive to targeted therapy agents that help it grow and invade healthy tissue. Targeted therapies for pancreatic cancer are a relatively new type of cancer treatment that targets specific molecules in the cancer cells. Important clinical progresses in diagnostic studies, surgical-techniques, and associated therapeutic strategies are sure to enhance survival of patients with pancreatic cancers. The better understanding of molecular manifestations involved in the pancreatic cancer and its progression can be helpful in developing promising therapeutic approaches.
Author Contributions: Conceptualization, L.K. and S.K.S.P.; data curation, L.K., S.K., K.S. and S.K.S.P.; writing-original draft preparation, L.K., S.K., K.S. and S.K.S.P.; writing-review and editing, L.K. and S.K.S.P. All authors have read and agreed to the published version of the manuscript.