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.

Prkci Regulates Autophagy and Pancreatic Tumorigenesis in Mice.

Protein kinase C iota (PKCι) functions as a bonafide human oncogene in lung and ovarian cancer and is required for KrasG12D-mediated lung cancer initiation and progression. PKCι expression is required for pancreatic cancer cell growth and maintenance of the transformed phenotype; however, nothing is known about the role of PKCι in pancreas development or pancreatic tumorigenesis. In this study, we investigated the effect of pancreas-specific ablation of PKCι expression on pancreatic cellular homeostasis, susceptibility to pancreatitis, and KrasG12D-mediated pancreatic cancer development. Knockout of pancreatic Prkci significantly increased pancreatic immune cell infiltration, acinar cell DNA damage, and apoptosis, but reduced sensitivity to caerulein-induced pancreatitis. Prkci-ablated pancreatic acinar cells exhibited P62 aggregation and a loss of autophagic vesicles. Loss of pancreatic Prkci promoted KrasG12D-mediated pancreatic intraepithelial neoplasia formation but blocked progression to adenocarcinoma, consistent with disruption of autophagy. Our results reveal a novel promotive role for PKCι in pancreatic epithelial cell autophagy and pancreatic cancer progression.

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.

Recurrent copy number gains drive PKCι expression and PKCι-dependent oncogenic signaling in human cancers.

PRKCI is frequently overexpressed in multiple human cancers, and PKCι expression is often prognostic for poor patient survival, indicating that elevated PKCι broadly plays an oncogenic role in the cancer phenotype. PKCι drives multiple oncogenic signaling pathways involved in transformed growth, and transgenic mouse models have revealed that PKCι is a critical oncogenic driver in both lung and ovarian cancers. We now report that recurrent 3q26 copy number gain (CNG) is the predominant genetic driver of PRKCI mRNA expression in all major human cancer types exhibiting such CNGs. In addition to PRKCI, CNG at 3q26 leads to coordinate CNGs of ECT2 and SOX2, two additional 3q26 genes that collaborate with PRKCI to drive oncogenic signaling and tumor initiation in lung squamous cell carcinoma. Interestingly however, whereas 3q26 CNG is a strong driver of PRKCI mRNA expression across all tumor types examined, it has differential effects on ECT2 and SOX2 mRNA expression. In some tumors types, particularly those with squamous histology, all three 3q26 oncogenes are coordinately overexpressed as a consequence of 3q26 CNG, whereas in other cancers only PRKCI and ECT2 mRNA are coordinately overexpressed. This distinct pattern of expression of 3q26 genes corresponds to differences in genomic signatures reflective of activation of specific PKCι oncogenic signaling pathways. In addition to highly prevalent CNG, some tumor types exhibit monoallelic loss of PRKCI. Interestingly, many tumors harboring monoallelic loss of PRKCI express significantly lower PRKCI mRNA and exhibit evidence of WNT/ß-catenin signaling pathway activation, which we previously characterized as a major oncogenic pathway in a newly described, PKCι-independent molecular subtype of lung adenocarcinoma. Finally, we show that CNG-driven activation of PKCι oncogenic signaling predicts poor patient survival in many major cancer types. We conclude that CNG and monoallelic loss are the major determinants of tumor PRKCI mRNA expression across virtually all tumor types, but that tumor-type specific mechanisms determine whether these copy number alterations also drive expression of the collaborating 3q26 oncogenes ECT2 and SOX2, and the oncogenic PKCι signaling pathways activated through the collaborative action of these genes. Our analysis may be useful in identifying tumor-specific predictive biomarkers and effective PKCι-targeted therapeutic strategies in the multitude of human cancers harboring genetic activation of PRKCI.

Protein kinase Cι promotes UBF1-ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of non-small-cell lung cancer cells.

Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinase Cι (PKCι) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.

Chromosome 3q26 Gain Is an Early Event Driving Coordinated Overexpression of the PRKCI, SOX2, and ECT2 Oncogenes in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (LSCC) is a prevalent form of lung cancer exhibiting distinctive histological and genetic characteristics. Chromosome 3q26 copy number gain (CNG) is a genetic hallmark of LSCC present in >90% of tumors. We report that 3q26 CNGs occur early in LSCC tumorigenesis, persist during tumor progression, and drive coordinate overexpression of PRKCI, SOX2, and ECT2. Overexpression of PRKCI, SOX2, and ECT2 in the context of Trp53 loss is sufficient to transform mouse lung basal stem cells into tumors with histological and genomic features of LSCC. Functionally, PRKCI and SOX2 collaborate to activate an extensive transcriptional program that enforces a lineage-restricted LSCC phenotype, whereas PRKCI and ECT2 collaborate to promote oncogenic growth. Gene signatures indicative of PKCι-SOX2 and PKCι-ECT2 signaling activity are enriched in the classical subtype of human LSCC and predict distinct therapeutic vulnerabilities. Thus, the PRKCI, SOX2, and ECT2 oncogenes represent a multigenic driver of LSCC.

Oncogenic protein kinase Cι signaling mechanisms in lung cancer: Implications for improved therapeutic strategies.

Protein Kinase Cι (PKCι) is a major oncogene involved in the initiation, maintenance and progression of numerous forms of human cancer. In the lung, PKCι is necessary for the maintenance of the transformed phenotype of the two major forms of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC). In addition, PKCι is necessary for both LADC and LSCC tumorigenesis by establishing and maintaining a highly aggressive stem-like, tumor-initiating cell phenotype. Interestingly however, while PKCι signaling in these two major lung cancer subtypes shares some common elements, it also drives distinct, sub-type specific pathways. Furthermore, recent analysis has revealed both PKCι-dependent and PKCι-independent pathways to LADC development. Herein, we discussion our current knowledge of oncogenic PKCι signaling in LADC and LSCC, and discuss these findings in the context of how they may inform strategies for improved therapeutic intervention in these deadly diseases.

Protein Kinase Cι and Wnt/ß-Catenin Signaling: Alternative Pathways to Kras/Trp53-Driven Lung Adenocarcinoma.

We report that mouse LSL-KrasG12D;Trp53fl/fl (KP)-mediated lung adenocarcinoma (LADC) tumorigenesis can proceed through both PKCι-dependent and PKCι-independent pathways. The predominant pathway involves PKCι-dependent transformation of bronchoalveolar stem cells (BASCs). However, KP mice harboring conditional knock out Prkci alleles (KPI mice) develop LADC tumors through PKCι-independent transformation of Axin2+ alveolar type 2 (AT2) stem cells. Transformed growth of KPI, but not KP, tumors is blocked by Wnt pathway inhibition in vitro and in vivo. Furthermore, a KPI-derived genomic signature predicts sensitivity of human LADC cells to Wnt inhibition, and identifies a distinct subset of primary LADC tumors exhibiting a KPI-like genotype. Thus, LADC can develop through both PKCι-dependent and PKCι-independent pathways, resulting in tumors exhibiting distinct oncogenic signaling and pharmacologic vulnerabilities.

Oncogenic Ect2 signaling regulates rRNA synthesis in NSCLC.

The Rho GTPase family members Rac1, Cdc42 and RhoA play key contributory roles in the transformed phenotype of human cancers. Epithelial Cell Transforming Sequence 2 (Ect2), a guanine nucleotide exchange factor (GEF) for these Rho GTPases, has also been implicated in a variety of human cancers. We have shown that Ect2 is frequently overexpressed in both major forms of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC), which together make up approximately 70% of all lung cancer diagnoses. Furthermore, we have found that Ect2 is required for multiple aspects of the transformed phenotype of NSCLC cells including transformed growth and invasion in vitro and tumorigenesis in vivo. More recently, we showed that a major mechanism by which Ect2 drives KRAS-mediated LADC transformation is by regulating rRNA (rRNA) synthesis. However, it remains unclear whether Ect2 plays a similar role in ribosome biogenesis in LSCC. Here we demonstrate that Ect2 expression correlates positively with expression of ribosome biogenesis genes and with pre-ribosomal 45S RNA abundance in primary LSCC tumors. Furthermore, we demonstrate that Ect2 functionally regulates rRNA synthesis in LSCC cells. Based on these data, we propose that inhibition of Ect2-mediated nucleolar signaling holds promise as a potential therapeutic strategy for improved treatment of both LADC and LSCC.

Protein kinase Cι: A versatile oncogene in the lung.

We have recently demonstrated that protein kinase Cι (PKCι) promotes a stem-like, tumor-initiating cell phenotype in KRAS-driven lung adenocarcinoma by activating a novel ELF3-NOTCH3 signaling axis.1 Combined PKCι and NOTCH inhibition was identified as a novel strategy for the treatment of KRAS-driven lung adenocarcinoma.

Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53-Driven Lung Adenocarcinoma.

The guanine nucleotide exchange factor (GEF) epithelial cell transforming sequence 2 (Ect2) has been implicated in cancer. However, it is not clear how Ect2 causes transformation and whether Ect2 is necessary for tumorigenesis in vivo. Here, we demonstrate that nuclear Ect2 GEF activity is required for Kras-Trp53 lung tumorigenesis in vivo and that Ect2-mediated transformation requires Ect2-dependent rDNA transcription. Ect2 activates rRNA synthesis by binding the nucleolar transcription factor upstream binding factor 1 (UBF1) on rDNA promoters and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA. Protein kinase Cι (PKCι)-mediated Ect2 phosphorylation stimulates Ect2-dependent rDNA transcription. Thus, Ect2 regulates rRNA synthesis through a PKCι-Ect2-Rac1-NPM signaling axis that is required for lung tumorigenesis.

Targeting oncogenic protein kinase Cι for treatment of mutant KRAS LADC.

Lung cancer is the leading cause of cancer death in the US with ∼124,000 new cases annually, and a 5 y survival rate of ∼16%. Mutant KRAS-driven lung adenocarcinoma (KRAS LADC) is a particularly prevalent and deadly form of lung cancer. Protein kinase Cι (PKCι) is an oncogenic effector of KRAS that activates multiple signaling pathways that stimulate transformed growth and invasion, and maintain a KRAS LADC tumor-initiating cell (TIC) phenotype. PKCι inhibitors used alone and in strategic combination show promise as new therapeutic approaches to treatment of KRAS LADC. These novel drug combinations may improve clinical management of KRAS LADC.

SOX2 Determines Lineage Restriction: Modeling Lung Squamous Cell Carcinoma in the Mouse.

In this issue of Cancer Cell, Ferone et al. demonstrate that SOX2 not only drives lung tumor formation but also restricts tumor lineage to squamous cell carcinoma (LSCC), regardless of cell of origin. This novel LSCC model should facilitate identification of key oncogenic drivers and treatment strategies for this lung cancer subtype.

Protein Kinase Cι Drives a NOTCH3-dependent Stem-like Phenotype in Mutant KRAS Lung Adenocarcinoma.

We report that the protein kinase Cι (PKCι) oncogene controls expression of NOTCH3, a key driver of stemness, in KRAS-mediated lung adenocarcinoma (LADC). PKCι activates NOTCH3 expression by phosphorylating the ELF3 transcription factor and driving ELF3 occupancy on the NOTCH3 promoter. PKCι-ELF3-NOTCH3 signaling controls the tumor-initiating cell phenotype by regulating asymmetric cell division, a process necessary for tumor initiation and maintenance. Primary LADC tumors exhibit PKCι-ELF3-NOTCH3 signaling, and combined pharmacologic blockade of PKCι and NOTCH synergistically inhibits tumorigenic behavior in vitro and LADC growth in vivo demonstrating the therapeutic potential of PKCι-ELF3-NOTCH3 signal inhibition to more effectively treat KRAS LADC.

The chromosome 3q26 OncCassette: A multigenic driver of human cancer.

Recurrent copy number variations (CNVs) are genetic alterations commonly observed in human tumors. One of the most frequent CNVs in human tumors involves copy number gains (CNGs) at chromosome 3q26, which is estimated to occur in >20% of human tumors. The high prevalence and frequent occurrence of 3q26 CNG suggest that it drives the biology of tumors harboring this genetic alteration. The chromosomal region subject to CNG (the 3q26 amplicon) spans from chromosome 3q26 to q29, a region containing ∼200 protein-encoding genes. The large number of genes within the amplicon makes it difficult to identify relevant oncogenic target(s). Whereas a number of genes in this region have been linked to the transformed phenotype, recent studies indicate a high level of cooperativity among a subset of frequently amplified 3q26 genes. Here we use a novel bioinformatics approach to identify potential driver genes within the recurrent 3q26 amplicon in lung squamous cell carcinoma (LSCC). Our analysis reveals a set of 35 3q26 amplicon genes that are coordinately amplified and overexpressed in human LSCC tumors, and that also map to a major LSCC susceptibility locus identified on mouse chromosome 3 that is syntenic with human chromosome 3q26. Pathway analysis reveals that 21 of these genes exist within a single predicted network module. Four 3q26 genes, SOX2, ECT2, PRKCI and PI3KCA occupy the hub of this network module and serve as nodal genes around which the network is organized. Integration of available genetic, genomic, biochemical and functional data demonstrates that SOX2, ECT2, PRKCI and PIK3CA are cooperating oncogenes that function within an integrated cell signaling network that drives a highly aggressive, stem-like phenotype in LSCC tumors harboring 3q26 amplification. Based on the high level of genomic, genetic, biochemical and functional integration amongst these 4 3q26 nodal genes, we propose that they are the key oncogenic targets of the 3q26 amplicon and together define a "3q26 OncCassette" that mediates 3q26 CNG-driven tumorigenesis. Genomic analysis indicates that the 3q26 OncCassette also operates in other major tumor types that exhibit frequent 3q26 CNGs, including head and neck squamous cell carcinoma (HNSCC), ovarian serous cancer and cervical cancer. Finally, we discuss how the 3q26 OncCassette represents a tractable target for development of novel therapeutic intervention strategies that hold promise for improving treatment of 3q26-driven cancers.

A small molecule inhibitor of atypical protein kinase C signaling inhibits pancreatic cancer cell transformed growth and invasion.

Pancreatic cancer is highly resistant to current chemotherapies. Identification of the critical signaling pathways that mediate pancreatic cancer transformed growth is necessary for the development of more effective therapeutic treatments. Recently, we demonstrated that protein kinase C iota (PKCι) and zeta (PKCζ) promote pancreatic cancer transformed growth and invasion, by activating Rac1→ERK and STAT3 signaling pathways, respectively. However, a key question is whether PKCι and PKCζ play redundant (or non-redundant) roles in pancreatic cancer cell transformed growth. Here we describe the novel observations that 1) PKCι and PKCζ are non-redundant in the context of the transformed growth of pancreatic cancer cells; 2) a gold-containing small molecule known to disrupt the PKCι/Par6 interaction, aurothiomalate, also disrupts PKCζ/Par6 interaction; 3) aurothiomalate inhibits downstream signaling of both PKCι and PKCζ, and blocks transformed growth of pancreatic cancer cells in vitro; and 4) aurothiomalate inhibits pancreatic cancer tumor growth and metastasis in vivo. Taken together, these data provide convincing evidence that an inhibitor of atypical PKC signaling inhibits two key oncogenic signaling pathways, driven non-redundantly by PKCι and PKCζ, to significantly reduce tumor growth and metastasis. Our results demonstrate that inhibition of atypical PKC signaling is a promising therapeutic strategy to treat pancreatic cancer.

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia.

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Complex role for the immune system in initiation and progression of pancreatic cancer.

The immune system plays a complex role in the development and progression of pancreatic cancer. Inflammation can promote the formation of premalignant lesions and accelerate pancreatic cancer development. Conversely, pancreatic cancer is characterized by an immunosuppressive environment, which is thought to promote tumor progression and invasion. Here we review the current literature describing the role of the immune response in the progressive development of pancreatic cancer, with a focus on the mechanisms that drive recruitment and activation of immune cells at the tumor site, and our current understanding of the function of the immune cell types at the tumor. Recent clinical and preclinical data are reviewed, detailing the involvement of the immune response in pancreatitis and pancreatic cancer, including the role of specific cytokines and implications for disease outcome. Acute pancreatitis is characterized by a predominantly innate immune response, while chronic pancreatitis elicits an immune response that involves both innate and adaptive immune cells, and often results in profound systemic immune-suppression. Pancreatic adenocarcinoma is characterized by marked immune dysfunction driven by immunosuppressive cell types, tumor-promoting immune cells, and defective or absent inflammatory cells. Recent studies reveal that immune cells interact with cancer stem cells and tumor stromal cells, and these interactions have an impact on development and progression of pancreatic ductal adenocarcinoma (PDAC). Finally, current PDAC therapies are reviewed and the potential for harnessing the actions of the immune response to assist in targeting pancreatic cancer using immunotherapy is discussed.