Ripk3 signaling regulates HSCs during stress and represses radiation-induced leukemia in mice.

Receptor-interacting protein kinase 3 (Ripk3) is one of the critical mediators of inflammatory cytokine-stimulated signaling. Here we show that Ripk3 signaling selectively regulates both the number and the function of hematopoietic stem cells (HSCs) during stress conditions. Ripk3 signaling is not required for normal homeostatic hematopoiesis. However, in response to serial transplantation, inactivation of Ripk3 signaling prevents stress-induced HSC exhaustion and functional HSC attenuation, while in response to fractionated low doses of ionizing radiation (IR), inactivation of Ripk3 signaling accelerates leukemia/lymphoma development. In both situations, Ripk3 signaling is primarily stimulated by tumor necrosis factor-α. Activated Ripk3 signaling promotes the elimination of HSCs during serial transplantation and pre-leukemia stem cells (pre-LSCs) during fractionated IR by inducing Mlkl-dependent necroptosis. Activated Ripk3 signaling also attenuates HSC functioning and represses a pre-LSC-to-LSC transformation by promoting Mlkl-independent senescence. Furthermore, we demonstrate that Ripk3 signaling induces senescence in HSCs and pre-LSCs by attenuating ISR-mediated mitochondrial quality control.

RIPK3 signaling and its role in the pathogenesis of cancers.

RIPK3 (receptor-interacting protein kinase 3) is a serine/threonine-protein kinase. As a key component of necrosomes, RIPK3 is an essential mediator of inflammatory factors (such as TNFα-tumor necrosis factor α) and infection-induced necroptosis, a programmed necrosis. In addition, RIPK3 signaling is also involved in the regulation of apoptosis, cytokine/chemokine production, mitochondrial metabolism, autophagy, and cell proliferation by interacting with and/or phosphorylating the critical regulators of the corresponding signaling pathways. Similar to apoptosis, RIPK3-signaling-mediated necroptosis is inactivated in most types of cancers, suggesting RIPK3 might play a critical suppressive role in the pathogenesis of cancers. However, in some inflammatory types of cancers, such as pancreatic cancers and colorectal cancers, RIPK3 signaling might promote cancer development by stimulating proliferation signaling in tumor cells and inducing an immunosuppressive response in the tumor environment. In this review, we summarize recent research progress in the regulators of RIPK3 signaling, and discuss the function of this pathway in the regulation of mixed lineage kinase domain-like (MLKL)-mediated necroptosis and MLKL-independent cellular behaviors. In addition, we deliberate the potential roles of RIPK3 signaling in the pathogenesis of different types of cancers and discuss the potential strategies for targeting this pathway in cancer therapy.

Targeting EphA2 suppresses hepatocellular carcinoma initiation and progression by dual inhibition of JAK1/STAT3 and AKT signaling.

Hepatocellular carcinoma (HCC) remains one of the deadliest malignancies worldwide. One major obstacle to treatment is a lack of effective molecular-targeted therapies. In this study, we find that EphA2 expression and signaling are enriched in human HCC and associated with poor prognosis. Loss of EphA2 suppresses the initiation and growth of HCC both in vitro and in vivo. Furthermore, CRISPR/CAS9-mediated EphA2 inhibition significantly delays tumor development in a genetically engineered murine model of HCC. Mechanistically, we discover that targeting EphA2 suppresses both AKT and JAK1/STAT3 signaling, two separate oncogenic pathways in HCC. We also identify a small molecule kinase inhibitor of EphA2 that suppresses tumor progression in a murine HCC model. Together, our results suggest EphA2 as a promising therapeutic target for HCC.

ABL1, Overexpressed in Hepatocellular Carcinomas, Regulates Expression of NOTCH1 and Promotes Development of Liver Tumors in Mice.

BACKGROUND & AIMS: We investigated whether ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) is involved in development of hepatocellular carcinoma (HCC). METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas. Albumin-Cre (HepWT) mice and mice with hepatocyte-specific disruption of Abl1 (HepAbl-/- mice) were given hydrodynamic injections of plasmids encoding the Sleeping Beauty transposase and transposons with the MET gene and a catenin ß1 gene with an N-terminal truncation, which induces development of liver tumors. Some mice were then gavaged with the ABL1 inhibitor nilotinib or vehicle (control) daily for 4 weeks. We knocked down ABL1 with short hairpin RNAs in Hep3B and Huh7 HCC cells and analyzed their proliferation and growth as xenograft tumors in mice. We performed RNA sequencing and gene set enrichment analysis of tumors. We knocked down or overexpressed NOTCH1 and MYC in HCC cells and analyzed proliferation. We measured levels of phosphorylated ABL1, MYC, and NOTCH1 by immunohistochemical analysis of an HCC tissue microarray. RESULTS: HCC tissues had higher levels of ABL1 than non-tumor liver tissues, which correlated with shorter survival times of patients. HepWT mice with the MET and catenin ß1 transposons developed liver tumors and survived a median 64 days; HepAbl-/- mice with these transposons developed tumors that were 50% smaller and survived a median 81 days. Knockdown of ABL1 in human HCC cells reduced proliferation, growth as xenograft tumors in mice, and expression of MYC, which reduced expression of NOTCH1. Knockdown of NOTCH1 or MYC in HCC cells significantly reduced cell growth. NOTCH1 or MYC overexpression in human HCC cells promoted proliferation and rescued the phenotype caused by ABL1 knockdown. The level of phosphorylated (activated) ABL1 correlated with levels of MYC and NOTCH1 in human HCC specimens. Nilotinib decreased expression of MYC and NOTCH1 in HCC cell lines, reduced the growth of xenograft tumors in mice, and slowed growth of liver tumors in mice with MET and catenin ß1 transposons, reducing tumor levels of MYC and NOTCH1. CONCLUSIONS: HCC samples have increased levels of ABL1 compared with nontumor liver tissues, and increased levels of ABL1 correlate with shorter survival times of patients. Loss or inhibition of ABL1 reduces proliferation of HCC cells and slows growth of liver tumors in mice. Inhibitors of ABL1 might be used for treatment of HCC.

Inhibition of insulin-like growth factor 1 receptor enhances the efficacy of sorafenib in inhibiting hepatocellular carcinoma cell growth and survival.

Hepatocellular carcinoma (HCC) is the fifth most common primary cancer and second largest cause of cancer-related death worldwide. The first-line oral chemotherapeutic agent sorafenib only increases survival in patients with advanced HCC by less than 3 months. Most patients with advanced HCC have shown limited response rates and survival benefits with sorafenib. Although sorafenib is an inhibitor of multiple kinases, including serine/threonine-protein kinase c-Raf, serine/threonine-protein kinase B-Raf, vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor ß, HCC cells are able to escape from sorafenib treatment using other pathways that the drug insufficiently inhibits. The aim of this study was to identify and target survival and proliferation pathways that enable HCC to escape the antitumor activity of sorafenib. We found that insulin-like growth factor 1 receptor (IGF1R) remains activated in HCC cells treated with sorafenib. Knockdown of IGF1R sensitizes HCC cells to sorafenib treatment and decreases protein kinase B (AKT) activation. Overexpression of constitutively activated AKT reverses the effect of knockdown of IGF1R in sensitizing HCC cells to treatment with sorafenib. Further, we found that ceritinib, a drug approved by the U.S. Food and Drug Administration for treatment of non-small cell lung cancer, effectively inhibits the IGF1R/AKT pathway and enhances the inhibitory efficacy of sorafenib in human HCC cell growth and survival in vitro, in a xenograft mouse model and in the c-Met/ß-catenin-driven HCC mouse model. Conclusion: Our study provides a biochemical basis for evaluation of a new combination treatment that includes IGF1R inhibitors, such as ceritinib and sorafenib, in patients with HCC. (Hepatology Communications 2018;2:732-746).

The role of Rak in the regulation of stability and function of BRCA1.

BRCA1 is an important player in the DNA damage response signaling, and its deficiency results in genomic instability. A complete loss or significantly reduced BRCA1 protein expression is often found in sporadic breast cancer cases despite the absence of genetic or epigenetic aberrations, suggesting the existence of other regulatory mechanisms controlling BRCA1 protein expression. Herein, we demonstrate that Fyn-related kinase (Frk)/Rak plays an important role in maintaining genomic stability, possibly in part through positively regulating BRCA1 protein stability and function via tyrosine phosphorylation on BRCA1 Tyr1552. In addition, Rak deficiency confers cellular sensitivity to DNA damaging agents and poly(ADP-ribose) polymerase (PARP) inhibitors. Overall, our findings highlight a critical role of Rak in the maintenance of genomic stability, at least in part, through protecting BRCA1 and provide novel treatment strategies for patients with breast tumors lacking Rak.

PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

Inhibition of SIRT2 suppresses hepatic fibrosis.

Liver fibrosis can progress to cirrhosis and result in serious complications of liver disease. The pathogenesis of liver fibrosis involves the activation of hepatic stellate cells (HSCs), the underlying mechanisms of which are not fully known. Emerging evidence suggests that the classic histone deacetylases play a role in liver fibrosis, but the role of another subfamily of histone deacetylases, the sirtuins, in the development of hepatic fibrosis remains unknown. In this study, we found that blocking the activity of sirtuin 2 (SIRT2) by using inhibitors or shRNAs significantly suppressed fibrogenic gene expression in HSCs. We further demonstrated that inhibition of SIRT2 results in the degradation of c-MYC, which is important for HSC activation. In addition, we discovered that inhibition of SIRT2 suppresses the phosphorylation of ERK, which is critical for the stabilization of c-MYC. Moreover, we found that Sirt2 deficiency attenuates the hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Furthermore, we showed that SIRT2, p-ERK, and c-MYC proteins are all overexpressed in human hepatic fibrotic tissues. These data suggest a critical role for the SIRT2/ERK/c-MYC axis in promoting hepatic fibrogenesis. Inhibition of the SIRT2/ERK/c-MYC axis represents a novel strategy to prevent and to potentially treat liver fibrosis and cirrhosis.

PKCα Attenuates Jagged-1-Mediated Notch Signaling in ErbB-2-Positive Breast Cancer to Reverse Trastuzumab Resistance.

PURPOSE: Breast cancer is the second leading cause of cancer mortality among women worldwide. The major problem with current treatments is tumor resistance, recurrence, and disease progression. ErbB-2-positive breast tumors are aggressive and frequently become resistant to trastuzumab or lapatinib. We showed previously that Notch-1 is required for trastuzumab resistance in ErbB-2-positive breast cancer. EXPERIMENTAL DESIGN: Here, we sought to elucidate mechanisms by which ErbB-2 attenuates Notch signaling and how this is reversed by trastuzumab or lapatinib. RESULTS: The current study elucidates a novel Notch inhibitory mechanism by which PKCα downstream of ErbB-2 (i) restricts the availability of Jagged-1 at the cell surface to transactivate Notch, (ii) restricts the critical interaction between Jagged-1 and Mindbomb-1, an E3 ligase that is required for Jagged-1 ubiquitinylation and subsequent Notch activation, (iii) reverses trastuzumab resistance in vivo, and (iv) predicts better outcome in women with ErbB-2-positive breast cancer. CONCLUSIONS: The clinical impact of these studies is PKCα is potentially a good prognostic marker for low Notch activity and increased trastuzumab sensitivity in ErbB-2-positive breast cancer. Moreover, women with ErbB-2-positive breast tumors expressing high Notch activation and low PKCα expression could be the best candidates for anti-Notch therapy.

Alcohol inhibits osteopontin-dependent transforming growth factor-ß1 expression in human mesenchymal stem cells.

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor ß1 (TGF-ß1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-ß1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-ß1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-ß1 protein expression, decreases MZF1-dependent TGF-ß1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-ß1 expression. Last, we showed that EtOH exposure reduces the TGF-ß1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-ß1 signaling pathway in MSC.

Targeting Fyn in Ras-transformed cells induces F-actin to promote adherens junction-mediated cell-cell adhesion.

Fyn, a member of the Src family kinases (SFK), is an oncogene in murine epidermis and is associated with cell-cell adhesion turnover and induction of cell migration. Additionally, Fyn upregulation has been reported in multiple tumor types, including cutaneous squamous cell carcinoma (cSCC). Introduction of active H-Ras(G12V) into the HaCaT human keratinocyte cell line resulted in upregulation of Fyn mRNA (200-fold) and protein, while expression of other SFKs remained unaltered. Transduction of active Ras or Fyn was sufficient to induce an epithelial-to-mesenchymal transition in HaCaT cells. Inhibition of Fyn activity, using siRNA or the clinical SFK inhibitor Dasatinib, increased cell-cell adhesion and rapidly (5-60 min) increased levels of cortical F-actin. Fyn inhibition with siRNA or Dasatinib also induced F-actin in MDA-MB-231 breast cancer cells, which have elevated Fyn. F-actin co-localized with adherens junction proteins, and Dasatinib-induced cell-cell adhesion could be blocked by Cytochalasin D, indicating that F-actin polymerization was a key initiator of cell-cell adhesion through the adherens junction. Conversely, inhibiting cell-cell adhesion with low Ca(2+) media did not block Dasatinib-induced F-actin polymerization. Inhibition of the Rho effector kinase ROCK blocked Dasatinib-induced F-actin and cell-cell adhesion, implicating relief of Rho GTPase inhibition as a mechanism of Dasatinib-induced cell-cell adhesion. Finally, topical Dasatinib treatment significantly reduced total tumor burden in the SKH1 mouse model of UV-induced skin carcinogenesis. Together these results identify the promotion of actin-based cell-cell adhesion as a newly described mechanism of action for Dasatinib and suggest that Fyn inhibition may be an effective therapeutic approach in treating cSCC.

FYNagling divergent adhesive functions for Fyn in keratinocytes.

Fyn, a member of the Src family kinases (SFKs), has been shown to play important yet contradictory roles in keratinocyte (KC) adhesion. During KC differentiation, physiological activation of Fyn results in the formation of adherens junctions, recruiting junctional components and inducing signaling pathways that control the differentiation program. However, in KC transformation and oncogenesis, increased Fyn activity has been implicated in the dissolution of adhesion structures and an increased migratory phenotype. Fyn activity is also associated with both the formation and dissolution of focal adhesions, and to a lesser extent hemidesmosomes and desmosomes. This viewpoint article aims to reconcile these disparate bodies of literature regarding Fyn's role in cell-cell and cell-matrix adhesion by proposing several alternative, testable hypotheses that unify Fyn's fractured functions.

BaxΔ2 promotes apoptosis through caspase-8 activation in microsatellite-unstable colon cancer.

UNLABELLED: Loss of apoptotic Bax due to microsatellite mutation contributes to tumor development and chemoresistance. Recently, a Bax microsatellite mutation was uncovered in combination with a specific alternative splicing event that could generate a unique Bax isoform (BaxΔ2) in otherwise Bax-negative cells. Like the prototype Baxα, BaxΔ2 is a potent proapoptotic molecule. However, the proapoptotic mechanism and therapeutic implication of BaxΔ2 remain elusive. Here, the isolation and analysis of isogenic subcell lines are described that represent different Bax microsatellite statuses from colorectal cancer. Colon cancer cells harboring Bax microsatellite G7/G7 alleles are capable of producing low levels of endogenous BaxΔ2 transcripts and proteins. Interestingly, BaxΔ2-positive cells are selectively sensitive to a subgroup of chemotherapeutics compared with BaxΔ2-negative cells. Unlike other Bax isoforms, BaxΔ2 recruits caspase-8 into the proximity for activation, and the latter, in turn, activates caspase-3 and apoptosis independent of the mitochondrial pathway. These data suggest that the expression of BaxΔ2 may provide alternative apoptotic and chemotherapeutic advantages for Bax-negative tumors. IMPLICATIONS: "Bax-negative" colorectal tumors expressing a Bax isoform are sensitive to selective chemotherapeutics.

Long QT2 mutation on the Kv11.1 ion channel inhibits current activity by ablating a protein kinase Cα consensus site.

Mutations that inhibit Kv11.1 ion channel activity contribute to abnormalities of cardiac repolarization that can lead to long QT2 (LQT2) cardiac arrhythmias and sudden death. However, for most of these mutations, nothing is known about the molecular mechanism linking Kv11.1 malfunction to cardiac death. We have previously demonstrated that disease-related mutations that create consensus sites for kinases on ion channels can dramatically change ion channel activity. Here, we show that a LQT2-associated mutation can inhibit Kv11.1 ion channel activity by perturbing a consensus site for the Ser/Thr protein kinase C α (PKCα). We first reveal by mass spectrometry analysis that Ser890 of the Kv11.1 ion channel is phosphorylated. Then, we demonstrate by a phospho-detection immunoassay combined with genetic manipulation that PKCα phosphorylates Ser890. Furthermore, we show that Ser890 phosphorylation is associated with an increase in Kv11.1 membrane density with alteration of recovery from inactivation. In addition, a newly discovered and as yet uncharacterized LQT2-associated nonsynonymous single nucleotide polymorphism 2660 G→A within the human ether-á-go-go-related gene 1 coding sequence, which replaces arginine 887 with a histidine residue (R887H), strongly inhibits PKCα-dependent phosphorylation of residue Ser890 on Kv11.1, and ultimately inhibits surface expression and current density. Taken together, our data provide a functional link between this channel mutation and LQT2.

Specifying protein kinase C functions in melanoma.

The protein kinase C (PKC) family of serine/threonine protein kinases is a heterogeneous group of enzymes receiving and integrating signals involved in both normal melanocyte biology and melanoma pathology. Alterations in PKC enzyme expression and activation contribute to the malignant phenotype of melanoma in both oncogenic and tumor suppressive roles. Delineating the diverse and often context-dependent functions of PKC enzymes in melanocyte/melanoma biology is key to capitalize on these kinases as drug targets. This review summarizes several of the diverse functions of PKC in melanocyte and melanoma biology with a focus on PKC enzyme regulation and function.

Fyn is induced by Ras/PI3K/Akt signaling and is required for enhanced invasion/migration.

Src family kinases (SFKs) are frequently over-expressed and/or activated in human cancers, and play key roles in cancer cell invasion, metastasis, proliferation, survival, and angiogenesis. Allosteric activation of SFKs occurs through well-defined post-translational mechanisms, however the SFK member Fyn is over-expressed in multiple human cancers (prostate, melanoma, pancreatic, glioma, chronic myelogenous leukemia) and the mechanism of increased Fyn expression is unclear. Since activation of Ras oncogenes is a common oncogenic event leading to the activation of multiple effector pathways, we explored if Ras could induce Fyn expression. Retroviral transduction of the human keratinocyte cell line HaCaT with oncogenic H-Ras dramatically up-regulated Fyn mRNA (>100-fold, P < 0.001), protein, and kinase activity without affecting Src levels or activity. Activation of Akt, but not MAPK or EGFR, was necessary and sufficient for induction of Fyn by H-Ras. Expression of active Fyn was sufficient to increase HaCaT cell migration and invasion, and the enhanced migration and invasion induced by H-Ras could be significantly blocked (70% reduction, P < 0.01) by knockdown of Fyn with a specific siRNA or inhibition of SFKs with PP2. In addition, expression of Fyn in MDA-MB-231 breast cancer cells was dependent on PI3K activity and was involved in their invasive phenotype. Thus, the Ras/PI3K/Akt pathway can account for Fyn over-expression in cancers, and Fyn is a critical mediator of the Ras-stimulated invasive cell phenotype. These results support the development of therapeutic strategies targeting Akt/Fyn pathway to block migration and invasion of tumor cells.

The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling.

Darier's disease (DD) is an inherited autosomal-dominant skin disorder characterized histologically by loss of adhesion between keratinocytes. DD is typically caused by mutations in sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), a major regulator of intracellular Ca(2+) homeostasis in the skin. However, a defined role for SERCA2 in regulating intercellular adhesion remains poorly understood. We found that diminution of SERCA2 function by pharmacological inhibition or siRNA silencing in multiple human epidermal-derived cell lines was sufficient to disrupt desmosome assembly and weaken intercellular adhesive strength. Specifically, SERCA2-deficient cells exhibited up to a 60% reduction in border translocation of desmoplakin (DP), the desmosomal cytolinker protein necessary for intermediate filament (IF) anchorage to sites of robust cell-cell adhesion. In addition, loss of SERCA2 impaired the membrane translocation of protein kinase C α (PKCα), a known regulator of DP-IF association and desmosome assembly, to the plasma membrane by up to 70%. Exogenous activation of PKCα in SERCA2-deficient cells was sufficient to rescue the defective DP localization, desmosome assembly, and intercellular adhesive strength to levels comparable to controls. Our findings indicate that SERCA2-deficiency is sufficient to impede desmosome assembly and weaken intercellular adhesive strength via a PKCα-dependent mechanism, implicating SERCA2 as a novel regulator of PKCα signaling.

Protein kinase C/mitogen-activated protein kinase signaling in keratinocyte differentiation control.

Proper epidermal keratinocyte differentiation, which is necessary for cutaneous barrier function, is altered in many common skin diseases. Keratinocyte differentiation is controlled by a complex signaling network involving multiple members of the protein kinase C and mitogen-activated protein kinase signaling kinases. Using an RNA interference knockdown approach, Adhikary et al. identified essential nodes in this signaling network, revealing remarkable kinase specificity.

Loss of protein kinase C delta gene expression in human squamous cell carcinomas: a laser capture microdissection study.

Protein kinase C delta (PKC-delta) protein levels are frequently low in chemically and UV-induced mouse skin tumors as well as in human cutaneous squamous cell carcinomas (SCCs). Furthermore, overexpression of PKC-delta in human SCC lines and mouse epidermis is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-delta a potential tumor suppressor gene for SCCs. Here we report that PKC-delta is lost in human SCCs at the transcriptional level. We used laser capture microdissection to isolate cells from three normal human epidermis and 14 human SCCs with low PKC-delta protein. Analysis by quantitative reverse transcription-PCR revealed that PKC-delta RNA was reduced an average of 90% in the SCCs tested, consistent with PKC-delta down-regulation at the protein level. Analysis of DNA from nine of the same tumors revealed that PKC-delta gene was deleted in only one tumor. In addition, Ras-transformed human keratinocytes, which have selective down-regulation of PKC-delta at both protein and mRNA levels, had significantly repressed human PKC-delta promoter activity. Together, these results indicate that PKC-delta gene expression is suppressed in human SCCs, probably via transcription repression. Our results have implications for the development of topical therapeutic strategies to trigger the re-expression of pro-apoptotic PKC-delta to induce apoptosis in SCCs.