PKCι Inhibition Research Articles

Abstract 4997: Auranofin enhances the efficacy of anti-PD1 immunotherapy

Abstract Anti-PD-1 immunotherapy brings hope for the treatment of malignant tumors, but its overall response rate is low. Therefore, how to sensitize anti-PD-1 therapy to make more patients benefit from immunotherapy is an urgent clinical demand. PKCι, a classical oncogene that affects cell proliferation and differentiation, has recently been found to be involved in the recruitment of immunosuppressive cells MDSCs and Tregs, which leads to anti-PD1 immunotherapy resistance. Our study is consistent with previously reported results that interfering with PKCι expression in tumor cells significantly enhances the anti-tumor effect of anti-PD1 therapy. Immunological mechanistic analyses demonstrated that the inhibition of PKCι expression significantly reduced the infiltration of tumor-associated macrophages (TAMs) rather than MDSCs or Tregs. Further studies revealed that PKCι enhanced the expression of macrophage chemokine CCL7 by promoting the formation of the YAP1/TEAD transcriptional complex. Interference in vitro and pharmacological inhibition of PKCι in tumor cells both significantly reduced CCL7 expression and TAMs invasion. Auranofin is a commonly used anti-inflammatory agent in clinical practice, which has been recently found that it directly induces tumor death by modulating the cellular redox system in tumor cells and remodels the tumor immunosuppressive microenvironment by specifically inhibiting PKCι expression. The results of our study showed that the anti-tumor effect of Auranofin combined with anti-PD1 therapy was superior to that of monotherapy in lung adenocarcinoma. Auranofin exerted synergistic anti-tumor effects with anti-PD1 immunotherapy by inhibiting the expression of PKCι-YAP1/TEAD-CCL7, reducing the recruitment of TAMs, and promoting the infiltration of effector CD8+ T cells. In summary, Auranofin is a potent candidate for sensitizing anti-PD-1 immunotherapy, reversing immunotherapy blunting by remodeling TIME, which provides new ideas and theoretical basis for immunotherapy of sensitizing malignant tumors. Citation Format: Zichen Zhao, Shichuan Hu, Yan Zhang. Auranofin enhances the efficacy of anti-PD1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4997.

Abstract 1672: PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma

Abstract Medulloblastoma is the second most common childhood cancer, behind leukemia, and the most common pediatric brain tumor, comprising 20% of all childhood brain cancers. Current treatment options are limited to radiation therapy, surgical resection, and chemotherapy, which are extremely invasive. This underscores the need for more targeted and effective therapeutic treatments to improve patient outcomes. The following study will focus on exploring the PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and its effects on medulloblastoma cell lines Daoy and ONS-76. Our in-vitro treatments showed that PKC-ι functions an oncogenic protein that is necessary for Daoy and ONS-76 cell proliferation and that ICA-1S was found to be cytostatic to both Daoy and ONS-76 cells. The inhibition of PKC-ι in both cell lines resulted in a decreased phosphorylation of PKC-ι as well as downregulation of 14-3-3 and crucial components of the c-Raf/Erk pathway including total and phosphorylated levels of c-Raf, Mek1/2, and Erk1/2. This comes as a result of ICA-1S blocking the association of PKC-ι with 14-3-3 while PKC-ι and c-Raf were not found to directly associate with each other, indicating indirect modulation of c-Raf by PKC-ι. Additionally, PKC-ι inhibition decreased its association with Stat3. Expression levels of Stat1, Stat3, c-Fos, and c-Myc transcription factors were significantly downregulated, which is likely the result of reduced transcriptional activation due to upstream inhibition of PKC-ι and Erk signaling. Treatment with ICA-1S was also shown to substantially decrease cellular invasion of Daoy and ONS-76 cells and induce apoptosis. Taken together, these data suggest that PKC-ι likely plays a critical role in the regulation of the c-Raf/Erk pathway, which controls the proliferation and invasion of human medulloblastoma. Citation Format: Luke Lajmi, Wishrawana S. Ratnayake, Joseph Palathinkal, Sloan Breedy, Aaron Todman, Mildred Acevedo-Duncan. PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1672.

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.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Abstract 5025: Reduced level of protein kinase C iota renders human glioblastoma less invasive and induces apoptosis via modulating Tak1/NF-κB pathway

Abstract Glioblastoma (GBM) is characterized by poor therapeutic response and overall survival. It is the most aggressive form of primary brain tumor with a tendency to invade surrounding healthy brain tissues, rendering them largely incurable. In this study, small-interference RNA was used to knock down the expression of protein kinase C PKC-ι. The reduced PKC-ι expression impaired the phosphorylation of LIM kinase 1/2 (LIMK), which is a critical step in cofilin recycling and actin polymerization. Additionally, there was a down-regulation of matrix metalloprotease-9 expression in LN-229 and U87G cells, which coincided with decreased invasion. Therefore, PKC-ι regulates both cytoskeleton rearrangement and cell adhesion, which contributed to cell migration. Silencing of PKC-ι also dampened Transforming growth factor-β (TGF-β)-activated kinase 1 (Tak1) level. TAK1 is an essential component in genotoxic stresses-induced NF-κB-activation and mitogen-activated protein kinase (MAPK)-pathways in GBM. TAK1, when activated, forms a complex with TAB1-3 (Tak1 binding proteins). Our data reveal, reduced Tak1 level after silencing PKC-ι interferes with complex formation and reduces NF-κB-activation. Diminished PKC-ι level also reduces activation of Tak1/JNK axis. This deactivation exerts an anti-oncogenic effect on glioblastoma via suppression of Akt1, c-Myc, and STAT3. This implies PKC-ι may have a crucial role in the activation of Tak1. Reduced migration and invasion in glioblastoma cells upon inhibition of PKC-ι was further confirmed by Scratch assay and Boyden assay. These results indicate that PKC-ι is involved in the control of glioblastoma cell migration and invasion. PKC-ι is a central component of multiple converging and bifurcating pathways and is a potential therapeutic target against GBM survival and infiltration. Citation Format: Khandker Mohammad Khalid, Wishrawana S. Ratnayake, Avijit Dey, Nuzhat N. Oishee, Mahfuza Marzan, Mildred Acevedo-Duncan. Reduced level of protein kinase C iota renders human glioblastoma less invasive and induces apoptosis via modulating Tak1/NF-κB pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5025.

Protein kinase Cι mediates immunosuppression in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most prevalent form of non-small cell lung cancer (NSCLC) and a leading cause of cancer death. Immune checkpoint inhibitors (ICIs) of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling induce tumor regressions in a subset of LUAD, but many LUAD tumors exhibit resistance to ICI therapy. Here, we identified Prkci as a major determinant of response to ICI in a syngeneic mouse model of oncogenic mutant Kras/Trp53 loss (KP)-driven LUAD. Protein kinase Cι (PKCι)-dependent KP tumors exhibited resistance to anti-PD-1 antibody therapy (α-PD-1), whereas KP tumors in which Prkci was genetically deleted (KPI tumors) were highly responsive. Prkci-dependent resistance to α-PD-1 was characterized by enhanced infiltration of myeloid-derived suppressor cells (MDSCs) and decreased infiltration of CD8+ T cells in response to α-PD-1. Mechanistically, Prkci regulated YAP1-dependent expression of Cxcl5, which served to attract MDSCs to KP tumors. The PKCι inhibitor auranofin inhibited KP tumor growth and sensitized these tumors to α-PD-1, whereas expression of either Prkci or its downstream effector Cxcl5 in KPI tumors induced intratumoral infiltration of MDSCs and resistance to α-PD-1. PRKCI expression in tumors of patients with LUAD correlated with genomic signatures indicative of high YAP1-mediated transcription, elevated MDSC infiltration and low CD8+ T cell infiltration, and with elevated CXCL5/6 expression. Last, PKCι-YAP1 signaling was a biomarker associated with poor response to ICI in patients with LUAD. Our data indicate that immunosuppressive PKCι-YAP1-CXCL5 signaling is a key determinant of response to ICI, and pharmacologic inhibition of PKCι may improve therapeutic response to ICI in patients with LUAD.

Protein kinase Cι and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.

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.

Regulation of a PRMT5/NF-κB Axis by Phosphorylation of PRMT5 at Serine 15 in Colorectal Cancer.

The overexpression of PRMT5 is highly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1β-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1β-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics.

Abstract 2857: Vimentin is a novel PKC-ι target facilitating migration and invasion in melanoma

Abstract Vimentin plays an important role in gaining rear-to-front polarity for mesenchymal cells, making it a hallmark of cancer metastasis. Epithelial-mesenchymal transition (EMT) is a vital process in tumor progression that converts stationary epithelial cells into motile mesenchymal phenotype by downregulating E-cadherin and upregulating Vimentin expression. Molecular dynamics of Vimentin intermediate filaments (VIFs) play several key roles in cancer metastasis. Vimentin is activated via phosphorylation through various kinases. Recently, we reported that Protein Kinase C-iota (PKC-ι), activates Vimentin in melanoma while inducing EMT. We also reported the effects of PKC-ι inhibition on mitigating EMT in melanoma based on PKC- ι specific inhibitors {Ratnayake, W.S., et al. Int. J. Oncol. 51(5), (2017), 1370-1382 https://doi.org/10.3892/ijo.2017.4131 and Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. This novel finding is very important with respect to cancer progression since PKC-ι is a well-established oncogene in various tumor types. Here, we analyzed the VIF dynamic changes upon PKC-ι inhibition using specific inhibitors targeting multiple activation sites on Vimentin. In addition to siRNA knockdown of expression of PKC-ι, we also report the effects of siRNA knockdown of two transcription factors, Paired related homeobox1 (PRRX1) and Zinc finger protein SNAI1 (SNAIL1) on the expressions of Vimentin, PKC-ι and E-cadherin. Herein we show that inhibition of PKC-ι downregulate the transcriptional activities of PRRX1 and SNAIL1, which was demonstrated with diminished levels of Vimentin and elevated levels of E-cadherin. Western blot, confocal immunofluorescence and immune-gold electron microscopic results suggested that PKC-ι targets multiple activation sites (S33, S39 and S56) on Vimentin without having an effect on S6 and S71 phosphorylation sites. Results also suggest that PKC-ι inhibition leads to diminution of activities of PRRX1 and SNAIL1 thereby further downregulation of Vimentin while upregulating E-cadherin in order to continue retardation of EMT in-vitro. Overall results indicate that PKC-ι is essential for VIF dynamics regulation during the metastasis in-vitro and therefore can be used as a novel biomarker to assess and mitigate melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Sloan Breedy, Christopher Apostolatos, Robert Hill, Clare Dennison, Mildred Acevedo-Duncan. Vimentin is a novel PKC-ι target facilitating migration and invasion in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2857.

Abstract 1044: PKC-ι and PKC-ζ are heavily responsible of up-regulating epithelial-mesenchymal transition (EMT) and activating Vimentin to facilitate cellular motility in prostate cancer cell lines

Abstract Prostate carcinoma is the most common cancer among men in terms of number of new cases reported each year. Approximately 26,000 deaths reported in 2018 in the United States and tumor metastasis is responsible for more than 90% of these deaths. Expression of Vimentin is a hallmark of metastatic mesenchymal prostate cancer cells which is initiated by EMT. Our previous studies show that atypical protein kinase C- iota (PKC-ι) and zeta (PKC-ζ) inhibition retards the activation of NF-κB pathway by diminishing NF-κB nuclei translocation {Apostolatos, A.H., et al. Int. J. Oncol. 53(5), (2018), 1836-1846 https://doi.org/10.3892/ijo.2018.4542}. The present study shows that siRNA knockdown of PKC-ι and PKC-ζ downregulate Snail1, PRRX1, Vimentin while upregulating E-cadherin thereby diminishes EMT. In-vitro migration and invasion assays for PC-3 and DU-145 prostate cancer cell lines demonstrated a significant reduction of cellular migration and invasion in PKC-ι and PKC-ζ knocked down samples. Immunoprecipitation experiments suggested direct association of Vimentin with PKC-ι and PKC-ζ separately. Laser stimulated confocal immunofluorescence and immuno-gold transmission electron microscopic techniques were used to further confirm the relationship of Vimentin with aPKCs. Real time qPCR was used to analyze the mRNA levels of targeted markers to further validate transcriptional downregulation of Vimentin which was observed in Westerns upon aPKC siRNA knockdown. Overall results showed stronger relationship between PKC-ι and Vimentin over PKC-ζ with Vimentin. Microscopic results also show PKC-ι concentrated along the cell membrane together with Vimentin in addition to the abundant distribution throughout the cell. In addition, our results suggest that both aPKCs target multiple activation sites (S33, S39 and S56) on Vimentin thereby playing a crucial role in the regulation of Vimentin dynamics which is essential for increased prostate cancer cell motility. We have used a novel PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) to conduct in-vivo experiments on murine models. Excised tumors were analyzed for pathways observed in in-vitro experiments. Immunohistochemical and Western blot analysis on tumor samples confirmed the relationship of aPKCs with Vimentin. In addition, these samples will be analyzed for the miRNA expression upon PKC-ι inhibition. Overall results suggest that both aPKCs are essential in upregulation of EMT and for the activation of Vimentin to facilitate metastasis of prostate cancer cells. Finally, results suggest that PKC-ι and PKC-ζ can be effectively targeted using specific inhibitors to develop targeted therapeutics for metastatic prostate carcinoma. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, Robert Hill, Clare Dennison, Mildred Acevedo-Duncan. PKC-ι and PKC-ζ are heavily responsible of up-regulating epithelial-mesenchymal transition (EMT) and activating Vimentin to facilitate cellular motility in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1044.

Abstract 5204: ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma

Abstract Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC- specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5204.

Abstract 2022: Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling

Abstract Melanoma is one of the fastest growing cancers in the United States, predicting a 14% increase in 2017 compared to 2016. The five year survival rate drops to 4% when melanoma is metastasized and responsible for >90% melanoma related deaths. Most available drugs target BRAF (V600E) mutation which occurs in ~60% melanoma cases, yet there is a poor prognosis and tumors acquire resistance to BRAF mutation inhibition. PKC-ι (iota) is an oncogene involved in cell cycle progression, tumorigenesis and cell survival in many cancers. We believe PKC-ι is an effective therapeutic target for invasive melanoma. We reported that PKC-ι is overexpressed in melanoma cells [Int. J. Oncol. 51(5), 1370-1382, (2017)]. In the current study, we have investigated the effects of novel PKC-ι inhibitor [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (1 µM) along with its nucleoside analog, 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (2.5 μM) on cell migration and invasion of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (MEL-NEO-F). Cell viability and WST-1 assays showed that both inhibitors show lesser cytotoxicity to MEL-F-NEO cell line at higher concentrations (>7.5 μM) compare to significant toxicity on melanoma (>1 μM). Both inhibitors decreased the levels of total and phosphorylated levels of PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and RhoA while decreasing the levels of Vimentin; a mesenchymal marker associated with EMT. Treatments with inhibitors significantly decreased the phosphorylated Vimentin (S39) and also altered the phosphorylation at S33 and S56, thereby preventing the Vimentin intermediate assembly. Immunoprecipitation and reversed immunoprecipitation showed a strong interaction of PKC-ι and Vimentin and immunofluorescence staining proved the observation. mRNA expression levels of PKC-ι, Vimentin decreased upon PKC-ι and the expression levels transcription factors such as SNAIL1, ZNF703, PRX1 will be tested using qPCR, Western blots and ELISA techniques. PKC-ι inhibition downregulate Par6 thereby stabilize RhoA even upon TGFβ1 stimulation. We have also showed that PKC-ι inhibition limits the translocation of activated NF-κB p65/p52 complex and β-catenin thereby regulates NF-κB and WNT/β-catenin signaling. Overall, results show that PKC-ι is essential for melanoma progression and metastasis through activation of Vimentin via TGFβ/Par6/RhoA pathway. Therefore PKC-ι could be used as effective therapeutic targets for malignant melanoma. Citation Format: Wishrawana S. Ratnayake, André H. Apostolatos, Christopher A. Apostolatos, Avijit Dey, Rekha Patel, Mildred Acevedo-Duncan. Oncogenic protein kinase Cι drives melanoma cell epithelial-mesenchymal transition by activating vimentin through Par6/RhoA signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2022.

Abstract 2542: c-Jun and FOXO1 play key roles in the overexpression of oncogenic PKC-ι in human prostate and melanoma cell lines

Abstract Protein Kinase C-ι (PKC-ι) is an anti-apoptotic oncogene overexpressed in multiple cancers including prostate, melanoma, ovarian, breast, and glioma. We have previously shown that PKC-ι is part of a cycle that helps cancer cells avoid senescence by releasing the transcription factor NF-kB. Additionally, PKC-ι has an anti-apoptotic effect while promoting epithelial-mesenchymal transition in melanoma {Ratnayake, et al. Int. J. Oncol. 51(5), (2017), 1370-1382}. PKC-ι is activated externally by factors like loss of PTEN and TGFβ stimulation. However treatment with PKC-ι specific inhibitors downregulate expression of both PKC-ι and phosphorylated PKC-ι, suggesting PKC-ι plays a role in regulating its own expression. A previous study showed the ELK1 transcription factor to be a regulator of PKC-ι (Gustafson, et al. J. Biol. Chem 279(10), (2003), 9400-8}. In this study, transcription factors c-Jun, ISGF3, PAX3, EGR1, and FOXO1 which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ι regulation in prostate cancer cell lines (DU145 and PC3) and melanoma cell lines (SK-MEL-2 and MeWo). Each transcription factor was systematically silenced. Western blots revealed that treatment with either c-Jun or FOXO1 siRNA significantly altered expression of PKC-ι. Results showed c-Jun followed by FOXO1 are the two major transcription factors which involve in PKC-ι expression in prostate cells compared to FOXO1 in melanoma cells. This suggests that these transcription factors act as activators that regulate PKC-ι expression and can be downstream targets of PKC-ι. We found that regulation of PKC-ι expression was governed mainly through NF-κB pathway. siRNA knockdown of NF-κB p65 and application of NF-κB inhibitors confirmed the above observation. We have also used immuno-paired-antibody detection assay to determine which pathways were affected with PKC-ι inhibitor treatments other than NF-κB pathway. qPCR and microarray were performed to analyze the transcriptome of treated cells to match protein levels with mRNA levels. We have analyzed multiple targets both up and downstream of PKC-ι to determine the mechanism of PKC-ι self-regulation. Overall results suggest that PKC-ι inhibition downregulates its own expression making it a novel target in prostate and melanoma cancer treatment. Citation Format: Andre H. Apostolatos, Wishrawana S. Ratnayake, Anisul Islam, Christopher Apostolatos, Tracess Smalley, Mildred Acevedo-Duncan. c-Jun and FOXO1 play key roles in the overexpression of oncogenic PKC-ι in human prostate and melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2542.

Abstract LB-B32: Modulation of the Hedgehog signaling pathway in models of basal cell carcinoma by ATP-competitive PKCi inhibitors

Abstract We will describe for the first time the optimization and pharmacological characterisation of azaquinazoline based ATP-competitive inhibitors of the aPKC isoforms. The serine/threonine kinase protein kinase C iota (PKCι) and protein kinase C zeta (PKCζ) together define the atypical sub-class of the protein kinase C superfamily (aPKC). Both isoforms have emerged as master regulators of cellular polarity with PKCι implicated in the maintenance of aberrant Hedgehog (HH) pathway activity in basal cell carcinoma (BCC). Common activating mutations in the HH signalling pathway drive BCC growth through the transcription factor GLI. Inhibitors of membrane protein smoothened (SMO) are clinically effective suppressors of HH signalling in BCC. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the clinical use of these agents. PKCι has been shown to directly activate GLI1 in BCC and has been linked to SMO inhibitor resistance making a strong case for the development of a selective PKCι inhibitor. Insights from structural biology identified critical amino acid residues in the PKCι binding pocket that could be exploited through structure-based drug design to optimise inhibitor potency and selectivity over structurally related kinase targets. The phosphorylation status of the known PKCι proximal substrate LLGL2 was used as a cellular pharmacodynamic biomarker and to demonstrate in vivo PKCι inhibition. To investigate the effects of PKCι inhibitors on the HH pathway a panel of compounds covering a diversity of chemotypes were profiled using a Hedgehog pathway GLI1 reporter assay in an engineered NIH3T3 cell line. Murine and human BCC cells, dependent on HH signalling for survival, were sensitive to PKCι inhibitor treatment with concomitant dose and time dependent effects on the expression of the HH pathway activity biomarker GLI1. Taken together, these results have therapeutic implications making a strong case for further evaluation of PKCι inhibitors in the setting of SMO-inhibitor-resistant BCC cancers. Citation Format: Jon Roffey, Christian Dillon, Anthony E. Oro, Amar N. Mirza, Kavita Y. Sarin, Sumaira Z. Aasi, Peter J. Parker, Philippe Riou, Caroline Barton, Bhavisha Patel, Caroline Barton, Andrew Turnbull, Emma Stanway, Katherine Fowler, Gregory Ott, Mark Ator. Modulation of the Hedgehog signaling pathway in models of basal cell carcinoma by ATP-competitive PKCi inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B32.

PKCι regulates nuclear YAP1 localization and ovarian cancer tumorigenesis.

Atypical protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancer. The PKCι gene PRKCI is targeted for frequent tumor-specific copy number gain (CNG) in both lung squamous cell carcinoma (LSCC) and ovarian serous carcinoma (OSC). We recently demonstrated that in LSCC cells PRKCI CNG functions to drive transformed growth and tumorigenicity by activating PKCι-dependent cell autonomous Hedgehog (Hh) signaling. Here, we assessed whether OSC cells harboring PRKCI CNG exhibit similar PKCι-dependent Hh signaling. Surprisingly, we find that whereas PKCι is required for the transformed growth for OSC cells harboring PRKCI CNG, these cells do not exhibit PKCι-dependent Hh signaling or Hh-dependent proliferation. Rather, transformed growth of OSC cells is regulated by PKCι-dependent nuclear localization of the oncogenic transcription factor, YAP1. Lentiviral shRNA-mediated knock down (KD) of PKCι leads to decreased nuclear YAP1 and increased YAP1 binding to angiomotin (AMOT), which sequesters YAP1 in the cytoplasm. Biochemical analysis reveals that PKCι directly phosphorylates AMOT at a unique site, Thr750, whose phosphorylation inhibits YAP1 binding. Pharmacologic inhibition of PKCι decreases YAP1 nuclear localization and blocks OSC tumor growth in vitro and in vivo. Immunohistochemical analysis reveals a strong positive correlation between tumor PKCι expression and nuclear YAP1 in primary OSC tumor samples, indicating the clinical relevance of PKCι-YAP1 signaling. Our results uncover a novel PKCι-AMOT-YAP1 signaling axis that promotes OSC tumor growth, and provide a rationale for therapeutic targeting of this pathway for treatment of OSC.

Abstract LB-052: Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification

Abstract Protein kinases are attractive targets for pharmacological intervention through their frequent disease associated aberration of cellular signalling networks. In this context an understanding of the myriad of substrates on which a protein kinase operates can help to reveal potential pharmacodynamic and patient stratification biomarkers. Kinase Identification of Proximal Substrates (KIPS) utilises an innovative chemical genetics approach to identify novel proximal biomarkers of kinase target response. This approach relies on the generation of a desensitized mutant protein that retains its kinase function, but loses sensitivity to small-molecule mediated inhibition. Comparative phospho-proteomics is performed on immuno-complexes of tagged wild type and mutant protein kinases in the presence and absence of inhibitor to isolate specific phospho-proteomic changes in proximal substrates. To exemplify the KIPS approach we have focused on the identification of novel substrates of the atypical protein kinase C isoform, PKCι. Through structure based design we identified two acidic amino acid residues in the PKCι nucleotide binding site that are crucial for efficacy of a previously characterized PKCι inhibitor, CRT0066854. Upon mutation to non-acidic alanine residues the PKCι protein is rendered insensitive to compound-mediated inhibition, allowing a comparison of wild type and mutant responses to PKCι inhibition to be made. Using HCT116 cells, we identified Myosin X as a putative substrate for PKCι, and demonstrated its specificity to the PKCι signalling axis through alanine-scanning peptide arrays and generation of phospho-specific antibodies. We show that the key acidic amino acids utilised in KIPS are highly conserved across the AGC and CAMK kinase super families. Furthermore, extensive compound SAR, diverse kinome profiling and structural biology have identified a tool box of inhibitor compounds with a broad range of activities across these kinase families leading to broader applicability of KIPS to enable the identification of individual kinase specific substrates and response across approximately 20% of the human kinome. Citation Format: jon roffey, Andrew Turnbull, Christian Dillon, Susan Boyd, Philippe Riou, Mark Linch, Peter Parker, Sven Kjaer, Neil McDonald. Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-052. doi:10.1158/1538-7445.AM2015-LB-052

Abstract A02: PKCι in pancreatic cancer development: A possible role for autophagy

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States with current 5-year survival rates at 6%. Greater than 90% of all pancreatic cancers harbor an activating mutation in codon 12 of the Kras gene (KrasG12D). Protein Kinase C iota (PKCι) has been shown to mediate KrasG12D downstream signaling and acinar-to-ductal metaplasia in an explant model of pancreatic cancer initiation. Additionally, inhibition of PKCι in human pancreatic cancer cells leads to decreased tumor size, angiogenesis, and metastasis in an orthotopic tumor model, suggesting that PKCι plays a role in maintaining the oncogenic phenotype of pancreatic cancer cells. We sought to test the hypothesis that PKCι plays a role development and maintenance of pancreatic cancer using a mouse model of KrasG12D-induced pancreatic cancer. Purpose: To identify the effect of PKCι loss on pancreas function and development of KrasG12D-induced pancreatic cancer. Procedures: Pancreata from mice with pancreas-specific deletion of PKCι, with and without pancreas-specific KrasG12D expression, were analyzed for histological and protein expression alterations. Acinar cells isolated from these transgenic mouse models were subjected to in vitro analyses to determine the role of PKCι in pancreatic cell homeostasis. Results: Mice lacking pancreatic PKCι survived and developed normally. Pancreatic knockout of PKCι expression resulted in a phenotype consistent with a defect in autophagic flux. In vitro analyses of isolated acinar cells supported the hypothesis that PKCι promotes autophagy in pancreatic cells. Autophagy exhibits a dual functionality in pancreatic cancer, acting as a tumor suppressor in normal cells and a tumor promoter in established tumor cells. We sought to determine the effect of PKCι knock-out on both of these cell types in a mouse model of pancreatic cancer. Using the LSL mouse model of K-ras induced pancreatic cancer, we demonstrated that pancreatic loss of PKCι lead to an increase in the number of Pancreatic Intraepithelial (PanIN) lesions formed in young mice. However, these lesions were protected from progressing past the early PanIN1/2 stage such that no mice lacking PKCι developed cancer, even after 1.5 years of age. By contrast, over 50% of mice with wild-type PKCι developed cancer; of those without cancer, many had progressed to the PanIN-3 stage, a stage representative of carcinoma in-situ. These data are similar to that seen in mouse models of pancreatic cancer with genetic inhibition of autophagy. Analysis of PanINs in these mice revealed that those lacking PKCι expressed significantly more P62 protein, a selective autophagy substrate, compared to PanINs with wild-type PKCι, supporting a block in the autophagic flux. These data suggest that PKCι plays an important role in both the initiation and progression of pancreatic cancer, possibly by regulating autophagic flux. Impact: Inhibition of pancreatic PKCι expression prevented progression of PanIN lesions past the early PanIN1/2 stage, resulting in a complete block in cancer development in mouse models of pancreatic cancer. Our preliminary data suggests that PKCι promotes cancer progression by regulating autophagic flux, a process that has been shown to be essential for KrasG12D-mediated pancreatic cancers. Pancreatic cancer is characterized by resistance to current therapies. Novel targets for new and more effective therapies are critical to improve outcomes for patients with this deadly disease. Inhibitors of PKCι are currently in clinical trials for ovarian and lung cancers. Our data supports the feasibility of using PKCι inhibitors for therapy in pancreatic cancer. Citation Format: Kristin S. Inman, Amanda A. Francis, Nicole R. Murray. PKCι in pancreatic cancer development: A possible role for autophagy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A02.

Atypical PKCι as target for glioblastoma therapy.

Glioblastoma (grade IV astrocytoma) is an aggressive and incurable form of brain tumor. It invariably shows extensive invasion at the time of diagnosis, often involving both hemispheres. Recent studies have given us a very detailed picture of glioblastoma genetics. These paint a picture of a disease with extensive heterogeneity, both between patients and within individual patients. This within patient heterogeneity presents a major challenge in the design of targeted therapies. One approach is to identify targets that are common downstream elements in signaling pathways that are aberrantly activated in glioblastoma. Here we review the evidence that the atypical protein kinase C family member PKCι may fulfill this role. Our current understanding of PKCι activation mechanisms is discussed and related to common genetic changes in glioblastoma. The data showing an essential role for PKCι in multiple aspects of glioblastoma pathology are also reviewed. Finally, data on the role of PKCι in normal brain function are reviewed for insights into potential side effects of PKCι inhibition in the central nervous system.

A Mixed-Methods Feasibility Trial of Protein Kinase C Iota Inhibition with Auranofin in Asymptomatic Ovarian Cancer Patients

Purpose: This trial was undertaken (1) to determine the feasibility of enrolling asymptomatic ovarian cancer patients with CA-125 elevation in a trial with the protein kinase C iota (PKCι) inhibitor auranofin and (2) to understand patients' perceptions of CA-125 monitoring. Methods: Asymptomatic ovarian cancer patients with CA-125 elevation received 3 mg auranofin orally twice per day and were evaluated. The patients participated in interviews about CA-125 monitoring. Results: Ten patients were enrolled in slightly over 6 months, exceeding our anticipated accrual rate. Four manifested stable CA-125 levels for 1 month or longer. The median progression-free survival was 2.8 months (95% CI: 1.3-3.8); auranofin was well tolerated. One patient had baseline and monthly CA-125 levels of 5,570, 6,085, 3,511, and 2,230 U/ml, respectively, stopped auranofin because of radiographic progression at 3 months, and manifested an increase in CA-125 to 7,168 U/ml approximately 3 months later. Patient interviews revealed (1) the important role of CA-125 in cancer monitoring, (2) ardent advocacy of CA-125 testing, and (3) an evolution toward CA-125 assuming a life of its own. Conclusions: This study showed the feasibility of enrolling asymptomatic ovarian cancer patients with CA-125 elevation in a trial with auranofin. One patient had a decline in CA-125, suggesting that PKCι inhibition merits further study in ovarian cancer. © 2014 S. Karger AG, Basel