The BAG3-IFITM2 Axis Enhances Pancreatic Ductal Adenocarcinoma Growth via the MAPK Signaling Pathway.

Pancreatic ductal adenocarcinoma (PDAC), a highly lethal malignancy, exhibits escalating incidence and mortality rates, underscoring the urgent need for the identification of novel therapeutic targets and strategies. The BAG3 protein, a multifunctional regulator involved in various cellular processes, notably plays a crucial role in promoting tumor progression and acts as a potential "bridge" between tumors and the tumor microenvironment. In this study, we demonstrate that PDAC cells secrete BAG3 (sBAG3), which engages the IFITM2 receptor to activate the MAPK signaling pathway, specifically enhancing pERK activity, thereby propelling PDAC growth. Furthermore, our preliminary investigation into the effects of sBAG3 on co-cultured NK cells intriguingly discovered that sBAG3 diminishes NK cell cytotoxicity and active molecule expression. In conclusion, our findings confirm the pivotal role of the sBAG3-IFITM2 axis in fostering PDAC progression, highlighting the potential significance of sBAG3 as a dual therapeutic target for both tumor and immune cells.

PKCι induces differential phosphorylation of STAT3 to modify STAT3-related signaling pathways in pancreatic cancer cells.

An increasing number of studies have documented atypical protein kinase C isoform ι (PKCι) as an oncoprotein playing multifaceted roles in pancreatic carcinogenesis, including sustaining the transformed growth, prohibiting apoptosis, strengthening invasiveness, facilitating autophagy, as well as promoting the immunosuppressive tumor microenvironment of pancreatic tumors. In this study, we present novel evidence that PKCι overexpression increases STAT3 phosphorylation at the Y705 residue while decreasing STAT3 phosphorylation at the S727 residue in pancreatic cancer cells. We further demonstrate that STAT3 phosphorylation at Y705 and S727 residues is mutually antagonistic, and that STAT3 Y705 phosphorylation is positively related to the transcriptional activity of STAT3 in pancreatic cancer cells. Furthermore, we discover that PKCι inhibition attenuates STAT3 transcriptional activity via Y705 dephosphorylation, which appears to be resulted from enhanced phosphorylation of S727 in pancreatic cancer cells. Finally, we investigate and prove that by modulating the STAT3 activity, the PKCι inhibitor can synergistically enhance the antitumor effects of pharmacological STAT3 inhibitors or reverse the anti-apoptotic side effects incited by the MEK inhibitor, thereby posing as a prospective sensitizer in the treatment of pancreatic cancer cells.

PKCι regulates the expression of PDL1 through multiple pathways to modulate immune suppression of pancreatic cancer cells.

To investigate the impact of oncogenic protein kinase C isoform ι (PKCι) on the microenvironment and the immunogenic properties of pancreatic tumors, we prohibit PKCι activity in various pancreatic ductal adenocarcinoma (PDAC) cell lines and co-culture them with human natural killer NK92 cells. The results demonstrate that PKCι suppression enhances the susceptibility of PDAC to NK cytotoxicity and promotes the degranulation and cytolytic activity of co-cultured NK92 cells. Mechanistic studies pinpoint that downstream of KRAS, both YAP1 and STAT3 are recruited by oncogenic PKCι to elevate the expression of PDL1, contributing to constitute an immune suppressive microenvironment in PDAC. Co-culture with NK92 further induces PDL1 upregulation via STAT3 to stimulate immune escape of PDAC cells. Subsequently, inhibition of PKCι in PDAC alleviates the immune suppression and enhances the cytotoxicity of NK92 towards PDAC through restraining PDL1 overexpression. Combined with PD1/PDL1 blocker, PKCι inhibitor remarkably elevates the cytotoxicity of NK92 against PDAC cells in vitro, establishing PKCι inhibitor as a promising candidate for boosting the immunotherapy of PDAC.

Transcription factor Sp1 is upregulated by PKCι to drive the expression of YAP1 during pancreatic carcinogenesis.

Recently, we identified that the atypical protein kinase C isoform ι (PKCι) enhances the expression of Yes-associated protein 1 (YAP1) to promote the tumorigenesis of pancreatic adenocarcinoma harboring mutant KRAS (mu-KRAS). To advance our understanding about underlying mechanisms, we analyze the transcription of YAP1 in pancreatic cancer cells and reveal that transcription factor specificity protein 1 (Sp1) is upregulated by PKCι and subsequently binds to multiple sites in YAP1 promoter to drive the transactivation of YAP1 in pancreatic cancer cells carrying mu-KRAS. The bioinformatics analysis further substantiates that the expression of PKCι, Sp1 and YAP1 is correlated and associated with the stages and prognosis of pancreatic tumors. Moreover, our apoptotic detection data demonstrate that combination of PKCι and Sp1 inhibitors at subtoxic doses displays synergistic effects on inducing apoptosis and reversing the immunosuppression of pancreatic cancer cells, establishing the combination of PKCι and Sp1 inhibitors as a promising novel therapeutic approach, or an adjuvant strategy to potentiate the antitumor effects of other immunotherapeutic agents in pancreatic cancer treatment.

Mu-KRAS attenuates Hippo signaling pathway through PKCι to sustain the growth of pancreatic cancer.

The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-mediated pancreatic tumorigenesis is not fully defined. In the present study, we demonstrate that mu-KRAS upregulates and activates PKCι, accompanied by dephosphorylation of large tumor suppressor (LATS), a key member of the growth-inhibiting Hippo signaling pathway. As a result, Yes-associated protein 1 (YAP1; a transcriptional coactivator) is dephosphorylated and translocates to the nucleus, which promotes transcription of downstream target genes to sustain the transformed growth of pancreatic cancer cells. In contrast, when PKCι is suppressed by the chemical inhibitor or small-hairpin RNA, the levels of phosphorylated LATS and YAP1 are elevated and YAP1 is excluded from the nucleus, which enhances the susceptibility of pancreatic cancer cells harboring mu-KRAS to apoptosis. These findings shed new light on the mechanisms underlying the pancreatic tumorigenesis initiated by mu-KRAS, and suggest that the PKCι-YAP1 signaling may potentially be therapeutically targeted for restricting the growth and inducing apoptosis in pancreatic tumors expressing mu-KRAS.

[The Effects of PKCι on Anti-tumor Activity of Cytokine-induced Killer Cells Against Pancreatic Cancer Cells and the Possible Underlying Mechanisms].

OBJECTIVE: To study the impact of atypical protein kinase Cι (PKCι) isoform PKC on the pancreatic cancer cells towards the tumoricidal effect of cytokine-induced killer (CIK) cells and explore its mechanisms. METHODS: CIK cells were prepared by inducing mononuclear cells isolated from the peripheral blood of healthy people with interleukin-2 (IL-2), interferon (IFN) and CD3 mAb and subsequently co-cultured with pancreatic epithelial cell HPDE6-C7, pancreatic cancer cells MiaPaCa and PANC-1 with or without PKC inhibitor named sodium thiomalate (ATM). All cells were divided into control group, ATM group, co-culture group with CIK and co-culture group with CIK+ATM. Cell count was used to detect the growth of each group from 1 to 8 d. Flow cytometry was used to detect the death rate of the cell lines after 48 h cell culture in each group. The small hairpin RNA (shRNA) was used for PKCι knockdown and the recombinant plasmid transfection was for PKCι overexpression in pancreatic cancer cells. Western blot and real-time fluorescent quantitative PCR (qRT-PCR) were utilized to determine the expression of PKCι protein and the impact on gene expression of transforming growth factor-ß (TGF-ß), a downstream effector modulated by PKC. Different mass concentrations of TGF-ß (1, 10, 20 ng/mL) were added into the co-culture of MiaPaCa and PANC-1 with CIK. The cell death rate was detected by flow cytometry 48 h later, so as to explore the possible mechanisms of the impact of PKCι on the tumoricidal effects of CIK cells. RESULTS: ATM and CIK were shown to suppress the growth and induce apoptosis or death of pancreatic cancer cells, meanwhile, ATM can enhance the tumoricidal effect of CIK on pancreatic cancer cells. Moreover, we found that PKCι knockdown in pancreatic cancer cells can down-regulate the gene expression of TGF-ß. In return, PKCι overexpression in pancreatic cancer cells can increase the gene expression of TGF-ß. The death rate of cancer cells with 10, 20 ng/mL TGF-ß was lower compared with the control group (P < 0.05). CONCLUSIONS: PKCι knockdown in pancreatic cancer cells can not only inhibit the growth of pancreatic cancer cells, but also enhance the tumoricidal effects of CIK on cancer cells. The possible mechanism of PKCι is to affect the immune escape of tumor cells by regulating the expression of TGF-ß.

PKCι and YAP1 are crucial in promoting pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant disease with 5-year survival rate of less than 6%. Activating mutations of Kras (mu-Kras) are often detected in most of PDAC patients. Although it has been known that oncogenic Kras is the driver of pancreatic cancer initiation and development, the underlying mechanisms by which mu-Kras promotes PDAC remain poorly understood. Here, we identify that PKCι is one of the crucial factors for supporting the survival of pancreatic cancer cells expressing mu-Kras. Our study demonstrates that after the knockdown of PKCι, the expression of the transcriptional co-activator YAP1 is decreased, which hinders the expression of the downstream target gene Mcl-1, and subsequently sensitizes pancreatic cancer MiaPaCa and PANC-1 cells experssing mu-Kras to apoptosis. In comparison, the suppression of PKCι has little impact on the viability of non-neoplastic pancreatic HPDE6-C7 cells. Moreover, the transient overexpression of oncogenic Kras in HPDE6-C7 elevates the expression of PKCι and YAP1 concomitantly. The upregulated YAP1 in HPDE6-C7/ mu-Kras cells is abolished once PKCι is suppressed, suggesting the linear relationship among mu-Kras, PKCι and YAP1. This phenomenon is further proven by the co-upregulation of PKCι and YAP1 in HPDE6-C7 cells stably transfected with mu-Kras. Taken together, our findings suggest that PKCι acts through promoting YAP1 function to promote the survival of pancreatic cancer cells expressing mu-Kras. It appears that targeting PKCι-YAP1 signaling is a feasible strategy for developing new therapeutics for treating pancreatic cancer patients.

[The Influence of New Medium with RGD on Cell Growth,Cell Fusion and Expression of Exogenous Gene].

OBJECTIVE: To investigate the influence of a new culture medium added with RGD on cell growth,cell fusion and expression of exogenous gene. METHODS: A new medium was prepared by adding different concentrations of RGD to ordinary culture medium. The optimum concentration of RGD was determined by observation of the growth of human pancreatic epithelial cell line HPDE6-C7. After determining the optimum concentration of RGD,different concentrations of cells HPDE6-C7 (5×104,105,5×105 mL－1) were inoculated in the two mediums. The morphology,adherence,growth and density of the cells were observed by inverted microscope; The ratio of clone formation and the positive rate of cloning were compared between the two cultures after fusion; The fluorescence intensity after the transfection of plasmid with green fluorescent protein (GFP) and the protein expression after transfection of plasmid with KRAS were observed to campare the expression of exogenous genes between the new medium with ordinary medium. RESULTS: Firstly,the optimal concentration of RGD was 10 ng/mL. Compared with the normal medium,the cultured cells with RGD had better morphology,adhesion and faster proliferation. In addition,both of the number and positive rate of clones formed in the new medium were significantly higher than that in the ordinary medium (P<0.05);The fluorescence intensity after transfection of exogenous gene GFP in the new medium was significantly higher than that in normal medium (P<0.05); Expression level of exogenous gene KRAS of the new medium was also significantly higher than that in normal medium. CONCLUSION: The new culture medium has highlighted advantages in cell growth,cell fusion and expression of exogenous genes. RGD peptide has widely prospect and potential value in the cell culture.

Muscarinic type 3 receptor induces cytoprotective signaling in salivary gland cells through epidermal growth factor receptor transactivation.

Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.

Nicotine overrides DNA damage-induced G1/S restriction in lung cells.

As an addictive substance, nicotine has been suggested to facilitate pro-survival activities (such as anchorage-independent growth or angiogenesis) and the establishment of drug resistance to anticancer therapy. Tobacco smoking consists of a variety of carcinogens [such as benzopyrene (BP) and nitrosamine derivatives] that are able to cause DNA double strand breaks. However, the effect of nicotine on DNA damage-induced checkpoint response induced by genotoxins remains unknown. In this study, we investigated the events occurred during G(1) arrest induced by γ-radiation or BP in nicotine-treated murine or human lung epithelial cells. DNA synthesis was rapidly inhibited after exposure to γ-radiation or BP treatment, accompanied with the activation of DNA damage checkpoint. When these cells were co-treated with nicotine, the growth restriction was compromised, manifested by upregulation of cyclin D and A, and attenuation of Chk2 phosphorylation. Knockdown of cyclin D or Chk2 by the siRNAs blocked nicotine-mediated effect on DNA damage checkpoint activation. However, nicotine treatment appeared to play no role in nocodazole-induced mitotic checkpoint activation. Overall, our study presented a novel observation, in which nicotine is able to override DNA damage checkpoint activated by tobacco-related carcinogen BP or γ-irradiation. The results not only indicates the potentially important role of nicotine in facilitating the establishment of genetic instability to promote lung tumorigenesis, but also warrants a dismal prognosis for cancer patients who are smokers, heavily exposed second-hand smokers or nicotine users.

Additive effects of orexin B and vasoactive intestinal polypeptide on LL-37-mediated antimicrobial activities.

The present study examined the bactericidal effects of orexin B (ORXB) and vasoactive intestinal peptide (VIP) alone or combined with cationic antimicrobial peptides, such as LL-37, on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus. The bactericidal effect of ORXB or VIP alone was detected in low NaCl concentration, but attenuated in physiological NaCl concentration (150 mM). However, such attenuated bactericidal activities of ORXB and VIP in 150 mM NaCl were regained by adding LL-37. Therefore, our results indicate that VIP and ORXB appear to mediate bactericidal effects in concert with LL-37 in the physiological context of mucosal tissue.

Differential sensitization of different prostate cancer cells to apoptosis.

Although protein kinase C (PKC) plays an important role in sensitizing prostate cancer cells to apoptosis, and suppression of PKC is able to trigger an apoptotic crisis in cells harboring oncogenic ras, little is known about whether dyregulation of Ras effectors in prostate cancer cells, together with loss of PKC, is synthetically lethal. The current study aims at investigating whether prostate cancer cells with aberrant Ras effector signaling are sensitive to treatment with HMG (a PKC inhibitor) for the induction of apoptosis. We show that prostate cancer DU145 cells expressing a high level of JNK1 become susceptible to apoptosis after treatment with HMG, in which caspase 8 is activated and cytochrome c is released to the cytosol. In contrast, the addition of HMG sensitizes LNCaP or PC3 prostate cancer cells harboring an active Akt to apoptosis, in which ROS is upregulated to induce the UPR and GADD153 expression. The concurrent activation of JNK1 and Akt has an additive effect on apoptosis following PKC suppression. Thus, the data identify Akt and JNK1 as potential targets in prostate cancer cells for PKC inhibition-induced apoptosis.

Synthetic Lethality Induced by Loss of PKC δ and Mutated Ras.

Synthetic lethal interaction between oncogenic Ha-ras and loss of PKC has been demonstrated. Recently, the authors reported that the concurrent knockdown of PKC α and ß, via upregulating PKC δ, sensitizes cells with aberrant Ras signaling to apoptosis. As a continuation of the study, using shRNA, the authors demonstrate that loss of PKC δ causes a lethal reaction in NIH3T3/Hras or prostate cancer DU145 cells that overexpress JNK. In this apoptotic process, PKC α and ß are upregulated and then associated with RACK1 (an adaptor for activated PKC) and JNK. Immunoblotting analysis shows that JNK is phosphorylated, accompanied with caspase 8 cleavage. The inhibition of JNK abrogates this apoptotic process triggered by PKC δ knockdown. Interestingly, without blocking PKC δ, the concurrent overexpression of wt- or CAT-PKC α and ß is insufficient to induce apoptosis in the cells. Together with the authors' previous findings, the data suggest that PKC α/ß and δ function oppositely to maintain a balance that supports cells expressing v-ras to survive and prevents them from being eliminated through oncogenic stress-induced apoptosis.

PI3K Acts in synergy with loss of PKC to elicit apoptosis via the UPR.

It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12-Ha-ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress-regulated apoptotic machinery.

Nicotine promotes mammary tumor migration via a signaling cascade involving protein kinase C and CDC42.

Nicotine, one of the major components in tobacco, is at high concentrations in the bloodstream of cigarette smokers. However, the mechanisms of how nicotine affects tumor development and whether nicotine is a potential carcinogen for malignancies induced by secondhand smoking are not fully understood yet. Here, we investigate the signaling pathways by which nicotine potentiates tumorigenesis in human mammary epithelial-like MCF10A or cancerous MCF7 cells. We show that human MCF10A and MCF7 cells both express four subunits of nicotine acetylcholine receptor (nAChR). The treatment of these cells with nicotine enhances the activity of protein kinase C (PKC) alpha without altering the expression level of this kinase. Nicotine also stimulates [(3)H]thymidine incorporation into the genome of these cells as well as forces serum-starved cells to enter S phase of the cell cycle, resulting in growth promotion. Importantly, on nicotine treatment, the mobility of MCF10A and MCF7 cells is enhanced, which can be blocked by the addition of nAChR or PKC inhibitor. Experiments using small interfering RNA knockdown or ectopic expression of cdc42 showed that cdc42 functions as a downstream effector of PKC and is crucial in the regulation of nicotine-mediated migratory activity in the cells. Together, our findings suggest that nicotine, through interacting with its receptor, initiates a signaling cascade that involves PKC and cdc42 and consequently promotes migration in mammary epithelial or tumor cells.

A medicinal mushroom: Phellinus linteus.

Phellinus Linteus (Berkeley & M. A. Curtis) Teng (PL) is a medicinal mushroom that has been practiced in oriental countries for centuries to prevent ailments as diverse as gastroenteric dysfunction, diarrhea, haemorrhage and cancers. In an effort to translate the Asian traditional medicines into western-accepted therapies, scientists have demonstrated that the extracts from fruit-bodies or mycelium of PL not only stimulate the hormonal and cell-mediated immune function and quench the inflammatory reactions caused by a variety of stimuli, but also suppress the tumor growth and metastasis. Mounting evidence from different research groups has shown that PL induces apoptosis in a host of murine and human carcinomas without causing any measurable toxic effects to their normal counterparts. Recently, research has been focused on the anti-tumor effect of PL, and in particular, on its ability to enhance some conventional chemotherapeutic drugs. These studies suggest PL to be a promising candidate as an alternative anticancer agent or a synergizer for existing antitumor drugs. Hereinafter, we summarize the present progress in elucidating the mechanisms underlying the potency of PL and its anti-tumor function. The fractionation and identification of the biologically active components from PL are also briefly introduced.

Modulation of intracellular signaling pathways to induce apoptosis in prostate cancer cells.

An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.

Modulation of lung cancer growth arrest and apoptosis by Phellinus Linteus.

The Phellinus Linteus (PL) mushroom has been shown to possess anti-tumor properties. Through influencing lymphocytes, PL indirectly augments the host's immune system against cancer cells. PL has also been demonstrated to reduce tumor proliferation. However, the mechanisms of PL against malignant growth have not yet been fully explored. In this study, we report that PL mediates the following two activities in mouse and human lung cancer cells: cell-cycle arrest at a low concentration of PL and apoptosis in response to a high dose of PL. After exposure to a low dose of PL, G(1) growth arrest occurred in the lung cancer cells. The negative growth control mediated by PL is evidenced by the decrease of the activities of cyclin-dependent kinases CDK2, 4, and 6. In contrast, at high doses, PL-induced lung cancer cells to undergo apoptosis in a dose-dependent fashion. This was evidenced by DNA fragmentation, caspase activation, and loss of clonogenecity in the lung cancer cells, all of which were lacking in the lung cancer cells treated with low concentrations of PL as well as the normal mouse lung epithelial cells exposed to either low or high concentrations of PL. The addition of the caspase inhibitor Z-VADfmk completely suppressed PL-induced apoptosis. Furthermore, the low dose of PL was able to synergize with doxorubicin to induce apoptosis in the lung cancer cells. Thus, our findings suggest that PL regulates two responses in the lung cancer cells: cell-cycle arrest and apoptosis.