PEGylation is a biochemical modification process of bioactive molecules with polyethylene glycol (PEG), which lends several desirable properties to proteins/peptides, antibodies, and vesicles considered to be used for therapy or genetic modification of cells. However, PEGylation of proteins is a complex process and can be carried out using more than one strategy that depends on the nature of the protein and the desired application. Proteins of interest are covalently conjugated or non-covalently complexed with inert PEG strings. Purification of PEGylated protein is another critical step, which is mainly carried out based on electrostatic interactions or molecular sizes using chromatography. Several PEGylated drugs are being used for diseases like anemia, kidney disease, multiple sclerosis, hemophilia and cancers. With the advancement and increased specificity of the PEGylation process, the world of drug therapy, and specifically cancer therapy could benefit by utilizing this technique to create more stable and nonimmunogenic therapies. In this article we describe the structure and functions of PEGylation and how this chemistry helps in drug discovery. Moreover, special emphasis has been given to CCN-family proteins that can be targeted or used as therapy to prevent or block cancer progression through PEGylation technology.

Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic-and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/ CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine in vitro and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabineresistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabineactivating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo. Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways.

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells' gemcitabine resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1 signaling plays a vital role in making pancreatic cancer cells resistant to gemcitabine i<i>n vitro</i> and also in a tumor xenograft model. We proved that the catastrophic effect of gemcitabine could significantly be increased in gemcitabine-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in gemcitabine-sensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, nontransformed pancreatic cells, which are sensitive to gemcitabine, cannot be resistant to gemcitabine by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN signaling that only influences PDAC cells to become resistant. Furthermore, we demonstrated that CYR61/CCN1 suppresses the expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells <i>in vitro</i> and <i>in vivo</i>. Thus, the previously described mechanisms (dCK and CTGF pathways) for gemcitabine resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from gemcitabine. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic gemcitabine resistance in PDAC cells by employing unique signaling pathways.</p></div>

<p>Fig. S1. Detection of cross-reactivity of Cyr61 and CTGF antibodies; Fig. S2. CYR61 ablation in AsPC-1 Cell Line using CRISPR/Cas9 Gene Editing; Fig. S3. Knockdown of CYR61 in Panc-1 cells and AsPC-1 cells using stable transfection of shRNA and CRISPR/Cas9 Gene Editing techniques, respectively; Fig. S4. CYR61 status in sub-populations (SP and NSP) in Panc-1 cells; Fig. S5. CYR61 suppresses the effect of Gemcitabine (GEM) on sphere-forming ability of Panc-1 cells; Fig. S6. Time-dependent effect of gemcitabine (GEM) on cell viability of three different PDAC cell lines; Fig. S7. Effect of hrCYR61/CCN1 on ð�›,-SMA expression in HPaSteC cells</p>

<p>Fig. S1. Detection of cross-reactivity of Cyr61 and CTGF antibodies; Fig. S2. CYR61 ablation in AsPC-1 Cell Line using CRISPR/Cas9 Gene Editing; Fig. S3. Knockdown of CYR61 in Panc-1 cells and AsPC-1 cells using stable transfection of shRNA and CRISPR/Cas9 Gene Editing techniques, respectively; Fig. S4. CYR61 status in sub-populations (SP and NSP) in Panc-1 cells; Fig. S5. CYR61 suppresses the effect of Gemcitabine (GEM) on sphere-forming ability of Panc-1 cells; Fig. S6. Time-dependent effect of gemcitabine (GEM) on cell viability of three different PDAC cell lines; Fig. S7. Effect of hrCYR61/CCN1 on ð�›,-SMA expression in HPaSteC cells</p>