P27Kip1 (CDKN1B) regulates cellular proliferation and senescence, and p27Kip1 deficiency in cancer is strongly correlated with poor prognosis of multiple cancer types. Understanding the mechanism of p27Kip1 loss in cancer and the consequences of restoring p27Kip1 levels is therefore critical for effective management during therapy. Here, SIRT1, a class III histone deacetylase (HDAC), is identified as an important regulator of p27Kip1 expression. Mechanistically, SIRT1 reduces p27Kip1 expression by decreasing p27Kip1 protein stability through the ubiquitin-proteasome pathway. In addition, SIRT1 silencing suppresses NSCLC proliferation and induces senescence in a p27Kip1-dependent manner. Furthermore, SIRT1 silencing dramatically suppresses tumor formation and proliferation in two distinct NSCLC xenograft mouse models. Collectively, these data not only demonstrate that SIRT1 is an important regulator of p27Kip1 but that SIRT inhibition induces senescence and anti-growth potential in lung cancer in vivo.
SIRT1 is a key regulator of p27 protein levels and SIRT1 inhibition is a viable strategy for NSCLC therapy by means of p27 reactivation.
SIRT1; p27kip1; protein stability; non-small cell lung cancer; cell senescence
Tobacco smoke contains multiple classes of established carcinogens including benzo(a)pyrenes, polycyclic aromatic hydrocarbons, and tobacco specific nitrosamines. Most of these compounds exert their genotoxic effects by forming DNA adducts and generation of reactive oxygen species, causing mutations in vital genes like K-Ras and p53. In addition, tobacco specific nitrosamines can activate nicotinic acetylcholine receptors (nAChRs) and to a certain extent β-Adrenergic receptors (β-ARs), promoting cell proliferation. Further, it has been demonstrated that nicotine, the major addictive component of tobacco smoke, can induce cell cycle progression, angiogenesis, and metastasis of lung and pancreatic cancers. These effects occur mainly through the α7-nAChRs, with possible contribution from the β-ARs and/or epidermal growth factor receptors (EGFRs). This review article will discuss the molecular mechanisms by which nicotine and its oncogenic derivatives such as NNK (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone) and NNN (N-nitrosonornicotine) induce cell cycle progression and promote tumor growth. A variety of signaling cascades are induced by nicotine through nAChRs, including the MAPK/ERK pathway, PI3K/AKT pathway and JAK/STAT signaling. In addition, studies have shown that nAChR activation induces Src kinase in a β-arrestin-1 dependent manner, leading to the inactivation of Rb protein and resulting in the expression of E2F1-regulated proliferative genes. Such nAChR-mediated signaling events enhance the proliferation of cells and render them resistant to apoptosis induced by various agents. These observations highlight the role of nAChRs in promoting the growth and metastasis of tumors and raise the possibility of targeting them for cancer therapy.
Experimental and population-based evidence has been steadily accumulating that steroid hormones are fundamentally involved in the biology of the lung. Both estrogen and progesterone receptors are present in normal and malignant lung tissue, and the reproductive hormones that bind these receptors have a role in lung development, lung inflammation, and lung cancer. The estrogen receptor β (ERβ) was discovered in the 1990's as a novel form of ER that is transcribed from a gene distinct from ERα, the receptor previously isolated from breast tissue. Interestingly, ERβ is the predominate ER expressed in normal and malignant lung tissue, while inflammatory cells that infiltrate the lung are known to express both ERα and ERβ. Although there is evidence from animal models for the preferential effects of ERβ in the lungs of females, human lung tumors from males often contain comparable numbers of ERβ-positive cells and male-derived lung cancer cell lines respond to estrogens. Lung tumors from both males and females also express CYP19 (aromatase), the rate-limiting enzyme in estrogen synthesis that converts testosterone to estrone and β-estradiol. Thus, testosterone acts as a precursor for local estrogen production within lung tumors, independent of reproductive organs. This review discusses the recent literature findings concerning the biology of the ERs, aromatase, and the progesterone receptor (PR) in lung cancer and highlights the ongoing clinical trials and future therapeutic implications of these findings.
Breast cancer malignancy is promoted by the small GTPases RhoA and RhoC. SmgGDS is a guanine nucleotide exchange factor that activates RhoA and RhoC in vitro. We previously reported that two splice variants of SmgGDS, SmgGDS-607 and SmgGDS-558, have different characteristics in binding and transport of small GTPases. To define the role of SmgGDS in breast cancer, we tested the expression of SmgGDS in breast tumors, and the role of each splice variant in proliferation, tumor growth, Rho activation, and NF-κB transcriptional activity in breast cancer cells. We show upregulated SmgGDS protein expression in breast cancer samples compared to normal breast tissue. Additionally, Kaplan-Meier survival curves indicated that patients with high SmgGDS expression in their tumors had worse clinical outcomes. Knockdown of SmgGDS-558, but not SmgGDS-607, in breast cancer cells decreased proliferation, in vivo tumor growth, and RhoA activity. Futhermore, we found that SmgGDS promoted a Rho-dependent activation of the transcription factor NF-κB, which provides a potential mechanism to define how SmgGDS-mediated activation of RhoA promotes breast cancer. This study demonstrates that elevated SmgGDS expression in breast tumors correlates with poor survival, and that SmgGDS-558 plays a functional role in breast cancer malignancy. Taken together, these findings define SmgGDS-558 as a unique promoter of RhoA and NF-κB activity and a novel therapeutic target in breast cancer.
SmgGDS; RhoA; breast cancer; NF-κB; Rap1GDS1
Macrophage migration inhibitory factor (MIF) is a homotrimeric proinflammatory cytokine implicated in chronic inflammatory diseases and malignancies, including cutaneous squamous cell carcinomas (SCC). To determine whether MIF inhibition could reduce UVB light–induced inflammation and squamous carcinogenesis, a small-molecule MIF inhibitor (CPSI-1306) was utilized that disrupts homotrimerization. To examine the effect of CPSI-1306 on acute UVB-induced skin changes, Skh-1 hairless mice were systemically treated with CPSI-1306 for 5 days before UVB exposure. In addition to decreasing skin thickness and myeloperoxidase (MPO) activity, CPSI-1306 pretreatment increased keratinocyte apoptosis and p53 expression, decreased proliferation and phosphohistone variant H2AX (γ-H2AX), and enhanced repair of cyclobutane pyrimidine dimers. To examine the effect of CPSI-1306 on squamous carcinogenesis, mice were exposed to UVB for 10 weeks, followed by CPSI-1306 treatment for 8 weeks. CPSI-1306 dramatically decreased the density of UVB-associated p53 foci in non–tumor-bearing skin while simultaneously decreasing the epidermal Ki67 proliferation index. In addition to slowing the rate of tumor development, CPSI-1306 decreased the average tumor burden per mouse. Although CPSI-1306–treated mice developed only papillomas, nearly a third of papillomas in vehicle-treated mice progressed to microinvasive SCC. Thus, MIF inhibition is a promising strategy for prevention of the deleterious cutaneous effects of acute and chronic UVB exposure.
We report that HMGN1, a nucleosome binding protein that affects chromatin structure and function, affects the growth of N-nitrosodiethylamine (DEN) induced liver tumors. Following a single DEN injection at 2 weeks of age, Hmgn1tm1/tm1 mice, lacking the nucleosome-binding domain of HMGN1, had earlier signs of liver tumorigenesis than their Hmgn1+/+ littermates. Detailed gene expression profiling revealed significant differences between DEN-injected and control saline injected mice, but only minor differences between the injected Hmgn1tm1/tm1 mice and their Hmgn1+/+ littermates. Pathway analysis revealed that the most significant process affected by loss of HMGN1 involves the lipid/ sterol metabolic pathway. Our study indicates that in mice, loss of HMGN1 leads to transcription changes that accelerate the progression of DEN-induced hepatocarcinogenesis, without affecting the type of tumors or the final total tumor burden of these mice.
This issue marks the 50th Anniversary of the release of the U.S. Surgeon General’s Report on Smoking and Health. Perhaps no other singular event has done more to highlight the effects of smoking on the development of cancer. Tobacco exposure is the leading cause of cancers involving the oral cavity, conductive airways and the lung. Owing to the many carcinogens in tobacco smoke, smoking-related malignancies have a high genome-wide burden of mutations, including in the gene encoding for p53. The p53 protein is the most frequently mutated tumor suppressor in cancer, responsible for a range of critical cellular functions that are compromised by the presence of a mutation. Herein we review the epidemiologic connection between tobacco exposure and cancer, the molecular basis of p53 mutation in lung cancer, and the normal molecular and cellular roles of p53 that are abrogated during lung tumor development and progression as defined by in vitro and in vivo studies. We also consider the therapeutic potential of targeting mutant p53 in a clinical setting based upon the cellular role of mutant p53 and data from genetic murine models.
Tobacco; Smoking; Surgeon General; Lung Cancer; p53; Metastasis; Animal models
Cancer stem cell characteristics, especially their self-renewal and clonogenic potentials, play an essential role in malignant progression and response to anti-cancer therapies. Currently, it remains largely unknown what pathways are involved in the regulation of cancer cell stemness and differentiation. Previously, we found that delta-like 1 homolog (Drosophila) or DLK1, a developmentally regulated gene, plays a critical role in regulation of differentiation, self-renewal, and tumorigenic growth of neuroblastoma cells. Here, we show that DLK1 specifically interacts with the prohibitin 1 (PHB1) and PHB2, two closely related genes with pleiotropic functions including regulation of mitochondrial function and gene transcription. DLK1 interacts with the PHB1-PHB2 complex via its cytoplasmic domain and regulates mitochondrial functions including mitochondrial membrane potential and production of reactive oxygen species (ROS). We have further found that PHB1 and especially PHB2 regulate cancer cell self-renewal as well as their clonogenic potential. Hence, the DLK1-PHB interaction constitutes a new signaling pathway that maintains clonogenicity and self-renewal potential of cancer cells.
cancer cell stemness; self-renewal; delta-like 1 homolog; DLK1; prohibitin; PHB
The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here, using a comprehensive breast cancer (BCa) tissue microarray, it was show that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly, it was shown that NEDD9 overexpression is a hallmark of highly invasive BCa cells. Moreover, NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo, leading to decreased circulating tumor cells (CTCs) and lung metastases in xenograft models. Mechanistically, NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Re-expression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of BCa cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of BCa cells.
This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis.
NEDD9; invasion, metastasis; breast cancer; MMP14
Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by ~70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1’s contribution to metastasis in this model. Re-expressing α3 in the α3-silenced cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3-silenced, and α3-rescued cells all secreted abundant laminin α5, an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and laminin-α5 are both over-expressed. Implications: α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin-α5 or other α3 integrin ligands are also over-expressed.
α3β1 integrin; laminin-332; laminin-511; breast cancer; metastasis
The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1 targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial ROS production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-kB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors.
Activated ALK and ROS1 tyrosine kinases, through gene fusions, has been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma (CRC) specimens using a 4-target, 4-color break-apart fluorescence in situ hybridization (FISH) assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical CRC specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intra-tumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in CRC.
ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.
Chemokines have been implicated as key contributors of non-small cell lung cancer (NSCLC) metastasis. However, the role of CXCR7, a recently discovered receptor for CXCL12 ligand, in the pathogenesis of NSCLC is unknown. To define the relative contribution of chemokine receptors to migration and metastasis we generated human lung A549 and H157 cell lines with stable knockdown of CXCR4, CXCR7, or both. Cancer cells exhibited chemotaxis to CXCL12 that was enhanced under hypoxic conditions, associated with a parallel induction of CXCR4, but not CXCR7. Interestingly, neither knockdown cell line differed in the rate of proliferation, apoptosis or cell adherence; however, in both cell lines, CXCL12-induced migration was abolished when CXCR4 signaling was abrogated. In contrast, inhibition of CXCR7 signaling did not alter cellular migration to CXCL12. In an in vivo heterotropic xenograft model using A549 cells, expression of CXCR4, but not CXCR7, on cancer cells was necessary for the development of metastases. In addition, cancer cells knocked-down for CXCR4 (or both CXCR4 and CXCR7) produced larger and more vascular tumors as compared to wild-type or CXCR7 knock-down tumors, an effect that was attributable to cancer cell-derived CXCR4 out competing endothelial cells for available CXCL12 in the tumor microenvironment. These results indicate that CXCR4, not CXCR7, expression engages CXCL12 to mediate NSCLC metastatic behavior.
bronchogenic carcinoma; CXCL12; angiogenesis; chemotaxis; A549; H157
Activating point mutations in the K-Ras oncogene are among the most common genetic alterations in pancreatic cancer, occurring early in the progression of the disease. However, the function of mutant K-Ras activity in tumor angiogenesis remains poorly understood. Using human pancreatic duct epithelial (HPDE) and K-Ras4BG12V–transformed HPDE (HPDE-KRas) cells, we show that activated K-Ras significantly enhanced the production of angiogenic factors including CXC chemokines and vascular endothelial growth factor (VEGF). Western blot analysis revealed that K-Ras activation promoted the phosphorylation of Raf/mitogen-activated protein kinase kinase-1/2 (MEK1/2) and expression of c-Jun. MEK1/2 inhibitors, U0126 and PD98059, significantly inhibited the secretion of both CXC chemokines and VEGF, whereas the c-Jun NH2-terminal kinase inhibitor SP600125 abrogated only CXC chemokine production. To further elucidate the biological functions of oncogenic K-Ras in promoting angiogenesis, we did in vitro invasion and tube formation assays using human umbilical vein endothelial cells (HUVEC). HUVEC cocultured with HPDE-KRas showed significantly enhanced invasiveness and tube formation as compared with either control (without coculture) or coculture with HPDE. Moreover, SB225002 (a CXCR2 inhibitor) and 2C3 (an anti-VEGF monoclonal antibody) either alone or in a cooperative manner significantly reduced the degree of both Ras-dependent HUVEC invasiveness and tube formation. Similar results were obtained using another pair of immortalized human pancreatic duct–derived cells, E6/E7/st and its oncogenic K-Ras variant, E6/E7/Ras/st. Taken together, our results suggest that angiogenesis is initiated by paracrine epithelial secretion of CXC chemokines and VEGF downstream of activated oncogenic K-Ras, and that this vascular maturation is in part dependent on MEK1/2 and c-Jun signaling.
Mutationally activated KRAS, detected in approximately 90% of pancreatic ductal adenocarcinomas (PDA), has proven an intractable pharmacologic target to date. Consequently, efforts to treat KRAS-mutated cancers are focused on targeting RAS-regulated signaling pathways. In mouse models, expression of BRAFV600E combined with dominant-negative TP53 elicits PDA, and pharmacologic blockade of mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibits proliferation of human PDA-derived cell lines. To better understand the role of RAF→MEK→ERK signaling on PDA cell proliferation, we assessed the consequences of MEK inhibition on global patterns of mRNA expression and tumor cell proliferation in a panel of human PDA-derived cell lines. This analysis revealed that RAF→MEK→ERK signaling regulates mRNAs involved in cell-cycle control as well as regulators of the immune system. Linear regression analysis of relative drug sensitivity and mRNA expression revealed mRNAs and pathways correlating with relative drug sensitivity of the cell lines. Mice carrying orthotopically implanted pancreas tumors that were treated with MEK inhibitor displayed reduced tumor growth, concomitant with a reduction of cells in S phase. Furthermore, analysis of tumor mRNA expression revealed PDA cell lines to display similar baseline and MEK inhibitor mRNA expression profiles in vitro and in vivo. Among the proteins subject to downregulation following MEK inhibition, we identified c-MYC as a key driver of cell proliferation downstream of RAF→MEK→ERK signaling. Indeed, in some PDA cell lines, RNA interference–mediated silencing of c-MYC expression had antiproliferative effects similar to that of MEK inhibition, thereby highlighting the importance of c-MYC in key aspects of pancreatic cancer cell maintenance.
Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs, including capacities for proliferation, self-renewal, and differentiation, resistance to chemotherapy, and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer.
Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling through inducing β-catenin degradation. Therefore, AR79, an inhibitor of GSK3β, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, β-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and β-catenin status, proliferation (with β-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-β-catenin expression and increased total and nuclear β-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation.
Wnt; glycogen synthase kinase 3β; prostate cancer; bone metastases; cancer stem cell
Chronic inflammation plays a significant role in tumor promotion, migration and invasion. Using microarray analysis, we observed a profound increase in genes involved in pro-inflammatory pathways in epidermal growth factor receptor inhibitor (EGFRI)-treated head and neck squamous cell carcinoma (HNSCC) cell lines compared to their respective vehicle-treated cell lines. We hypothesized that the efficacy of EGFRIs may be offset by the pro-inflammatory response that these drugs produce in HNSCC tumor cells. We found that clinical EGFRIs such as erlotinib, cetuximab, lapatinib and panitumumab induced the secretion of pro-inflammatory cytokines such as IL-2, IL-4, IL-6, IL-8, GM-CSF, TNFα and IFNγ. Focusing on IL-6, we found that erlotinib induced a time-dependent increase in IL-6 mRNA and protein expression and exogenous IL-6 was able to protect HNSCC cells from erlotinib-induced cytotoxicity. Conversely, an IL-6 receptor antagonist tocilizumab, sensitized HNSCC cells to erlotinib in vitro and in vivo. Inhibitors of NFκB, p38 and JNK suppressed erlotinib-induced IL-6 expression, suggesting an important role of NFκB and MAPK pathways in IL-6 expression. Furthermore, knockdown of NADPH oxidase 4 (NOX4) suppressed erlotinib-induced pro-inflammatory cytokines expression. Taken together, these results suggest that clinical EGFRIs induce the expression of pro-inflammatory cytokines via NOX4. Therefore, the anti-tumor activity of EGFRIs may be partially reduced by activation of NOX4-mediated pro-inflammatory pathways in HNSCC.
EGFR; Erlotinib; IL-6; inflammation; cytokines; NOX4; HNSCC
Ursolic acid (UA), present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to UA treatment. Herein, skin carcinoma cells (Ca3/7) as compared to skin papilloma cells (MT1/2) displayed more resistance to UA-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in pre-clinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate Rhodamine 123 (Rh123) indicating UA is transported by P-gp. To determine if P-gp inhibition can enhance UA-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil (VRP) or cyclosporin A (CsA). Alternatively, cells were pre-treated with the natural compound resveratrol (RES), a known chemotherapy sensitizer. VRP and RES enhanced the effects of UA in both cell lines, while CsA only did so in Ca3/7 cells. Similarly, VRP inhibited Rh123 efflux in both lines, while CsA only inhibited Rh123 efflux in Ca3/7 cells. RES did not inhibit Rh123 efflux in either line, indicating the synergistic effects of RES and UA are not manifest by inhibition of P-gp-mediated efflux of UA. These results indicate that the anti-skin cancer effects of UA are enhanced with P-gp inhibitors. In addition, RES and UA interact synergistically, but not through inhibition of P-gp.
Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen.
skin cancer; ursolic acid; p-glycoprotein; resveratrol; cytotoxicity
Protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancers. PKCι is an attractive therapeutic target for treatment of lung cancer, particularly those whose tumors express elevated PKCι. However, it is unknown whether PKCι is a viable therapeutic target in the ovary, and virtually nothing is known about the mechanism by which PKCι drives ovarian tumorigenesis. Here, we demonstrate that PKCι maintains a tumor-initiating cell (TIC) phenotype that drives ovarian tumorigenesis. A highly tumorigenic population of cells from human ovarian cancer cell lines exhibit properties of cancer stem-like TICs including self-renewal, clonal expansion, expression of stem-related genes, enhanced transformed growth in vitro, and aggressive tumor-initiating activity in vivo. Genetic disruption of PKCι inhibits the proliferation, clonal expansion, anchorage-independent growth and enhanced tumorigenic properties of ovarian TICs. Biochemical analysis demonstrates that PKCι acts through its oncogenic partner Ect2 to activate a Mek-Erk signaling axis that drives the ovarian TIC phenotype. Genomic analysis reveals that PRKCI and ECT2 are coordinately amplified and overexpressed in the majority of primary ovarian serous tumors, and these tumors exhibit evidence of an active PKCι-Ect2 signaling axis in vivo. Finally, we demonstrate that auranofin is a potent and selective inhibitor of oncogenic PKCι signaling that inhibits the tumorigenic properties of ovarian TIC cells in vitro and in vivo. Our data demonstrate that PKCι is required for a TIC phenotype in ovarian cancer, and that auranofin is an attractive therapeutic strategy to target deadly ovarian TICs in ovarian cancer patients.
Resistance to antiangiogenic therapies is a critical problem that has limited the utility of antiangiogenic agents in clinical settings. However, the molecular mechanisms underlying this resistance have yet to be fully elucidated. In this study, we established a novel xenograft model of acquired resistance to bevacizumab. To identify molecular changes initiated by the tumor cells, we performed human-specific microarray analysis on bevacizumab-sensitive and -resistant tumors. Efficiency analysis identified 150 genes upregulated and 31 genes downregulated in the resistant tumors. Among angiogenesis-related genes, we found upregulation of fibroblast growth factor-2 (FGF2) and fibroblast growth factor receptor-3 (FGFR3) in the resistant tumors. Inhibition of the FGFR in the resistant tumors led to the restoration of sensitivity to bevacizumab. Furthermore, increased FGF2 production in the resistant cells was found to be mediated by overexpression of upstream genes phospholipase C (PLCg2), frizzled receptor-4 (FZD4), chemokine [C-X3-C motif] (CX3CL1), and chemokine [C-C motif] ligand 5 (CCL5) via extracellular signal-regulated kinase (ERK). In summary, our work has identified an upregulation of a proangiogenic signature in bevacizumab-refractory HNSCC tumors that converges on ERK signaling to upregulate FGF, which then mediates evasion of anti-VEGF therapy. These findings provide a new strategy on how to enhance the therapeutic efficacy of antiangiogenic therapy.
Novel xenograft model leads to the discovery of FGF as a promising therapeutic target in overcoming the resistance of antiangiogenic therapy in HNSCC.
Acquired Resistance; Bevacizumab; Biomarker; FGF; HNSCC