ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

Case Report: Combined Intra-Lesional IL-2 and Topical Imiquimod Safely and Effectively Clears Multi-Focal, High Grade Cutaneous Squamous Cell Cancer in a Combined Liver and Kidney Transplant Patient.

Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin cancer worldwide, with ever increasing incidence and mortality. While most patients can be treated successfully with surgical excision, cryotherapy, or radiation therapy, there exist a subset of patients with aggressive cSCC who lack adequate therapies. Among these patients are solid organ transplant recipients who due to their immunosuppression, develop cSCC at a dramatically increased rate compared to the normal population. The enhanced ability of the tumor to effectively undergo immune escape in these patients leads to more aggressive tumors with a propensity to recur and metastasize. Herein, we present a case of aggressive, multi-focal cSCC in a double organ transplant recipient to frame our discussion and current understanding of the immunobiology of cSCC. We consider factors that contribute to the significantly increased incidence of cSCC in the context of immunosuppression in this patient population. Finally, we briefly review current literature describing experience with localized therapies for cSCC and present a strong argument and rationale for consideration of an IL-2 based intra-lesional treatment strategy for cSCC, particularly in this immunosuppressed patient population.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

Mast Cells and Skin and Breast Cancers: A Complicated and Microenvironment-Dependent Role.

Mast cells are important sentinel cells in host defense against infection and major effector cells in allergic disease. The role of these cells in cancer settings has been widely debated. The diverse range of mast cell functions in both immunity and tissue remodeling events, such as angiogenesis, provides multiple opportunities for mast cells to modify the tumor microenvironment. In this review, we consider both skin and breast cancer settings to address the controversy surrounding the importance of mast cells in the host response to tumors. We specifically address the key mediators produced by mast cells which impact tumor development. The role of environmental challenges in modifying mast cell responses and opportunities to modify mast cell responses to enhance anti-tumor immunity are also considered. While the mast cell's role in many cancer contexts is complicated and poorly understood, the activities of these tissue resident and radioresistant cells can provide important opportunities to enhance anti-cancer responses and limit cancer development.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Quantitative Proteome Responses to Oncolytic Reovirus in GM-CSF- and M-CSF-Differentiated Bone Marrow-Derived Cells.

The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.

HDAC6 differentially regulates autophagy in stem-like versus differentiated cancer cells.

Cancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

TAp73 Modifies Metabolism and Positively Regulates Growth of Cancer Stem-Like Cells in a Redox-Sensitive Manner.

PURPOSE: Stem-like cancer cells, with characteristic self-renewal abilities, remain highly refractory to various clinical interventions. As such, stemness-inhibiting entities, such as tumor suppressor p53, are therapeutically pursued for their anticancer activities. Interestingly, similar implications for tumor suppressor TAp73 in regulating stemness features within stem-like cancer cells remain unknown.Experimental Design: This study utilizes various in vitro molecular biology techniques, including immunoblotting, qRT-PCR, and mass spectrometry-based proteomics, and metabolomics approaches to study the role of TAp73 in human and murine embryonal carcinoma stem-like cells (ECSLC) as well as human breast cancer stem-like cells (BCSLC). These findings were confirmed using patient-derived brain tumor-initiating cells (BTIC) and in vivo xenograft models. RESULTS: TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in ECSLCs. In vivo, TAp73-deficient ECSLCs and BCSLCs demonstrate decreased tumorigenic potential when xenografted in mice. Mechanistically, TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, OAT, and PYCR1 involved in the interconversion of proline-glutamine-ornithine. Further, TAp73 deficiency exacerbates glutamine dependency, enhances accumulation of reactive oxygen species through reduced superoxide dismutase 1 (SOD1) expression, and promotes differentiation by arresting cell cycle and elevating autophagy. Most importantly, the knockdown of TAp73 in CD133HI BTICs, separated from three different glioblastoma patients, strongly decreases the expression of prosurvival factors Sox-2, BMI-1, and SOD1, and profoundly decreases their self-renewal capacity as evidenced through their reduced tumorsphere formation ability. CONCLUSIONS: Collectively, we reveal a clinically relevant aspect of cancer cell growth and stemness regulation through TAp73-mediated redox-sensitive metabolic reprogramming.

DNA Methylation Predicts the Response of Triple-Negative Breast Cancers to All-Trans Retinoic Acid.

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.

Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.

Aldehyde dehydrogenase 1A3 influences breast cancer progression via differential retinoic acid signaling.

Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

Malignant triton tumors--complete surgical resection and adjuvant radiotherapy associated with improved survival.

BACKGROUND: Malignant triton tumors (MTT) are a rare form of peripheral nerve sheath tumors that follows a particularly aggressive course. Given its rarity, only case reports and small series of patients have been published. METHODS: A Pubmed search was conducted (1966-2009) using the terms "triton tumor," "rhabdomyosarcoma," and "malignant peripheral nerve sheath tumor." The reference lists of retrieved articles were searched. Cases were included when the diagnosis was clear, the patient underwent surgery, and follow-up data were available. Univariate and multivariate analyses were conducted for predictors of positive resection margin, local recurrence/progression, development of metastases, and mortality. RESULTS: A total of 124 cases were included. The overall 5-year survival was 14% and the median time to death was 13 months. The overall local recurrence/progression rate was 50% and the median time to recurrence/progression was 6 months. On multivariate Cox proportional hazards analysis, positive margin status (HR 2.2, P = 0.01), local recurrence/progression (HR 3.1, P = 0.003), and development of metastases (HR 2.6, P = 0.003) were associated with mortality. Adjuvant radiotherapy was associated with improved survival (HR 0.4, P = 0.005). CONCLUSION: Complete surgical resection and adjuvant radiotherapy should be the cornerstones of treatment for MTT.

Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1, one of 19 ALDH isoforms expressed in humans, was generally believed to be responsible for the ALDH activity of CSCs. More recently, experiments with murine hematopoietic stem cells, murine progenitor pancreatic cells, and human breast CSCs indicate that other ALDH isoforms, particularly ALDH1A3, significantly contribute to aldefluor positivity, which may be tissue and cancer specific. Therefore, potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators.

Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis.

Cancer stem cells (CSCs) are proposed to initiate cancer and propagate metastasis. Breast CSCs identified by aldehyde dehydrogenase (ALDH) activity are highly tumorigenic in xenograft models. However, in patient breast tumor immunohistological studies, where CSCs are identified by expression of ALDH isoform ALDH1A1, CSC prevalence is not correlative with metastasis, raising some doubt as to the role of CSCs in cancer. We characterized the expression of all 19 ALDH isoforms in patient breast tumor CSCs and breast cancer cell lines by total genome microarray expression analysis, immunofluorescence protein expression studies, and quantitative polymerase chain reaction. These studies revealed that ALDH activity of patient breast tumor CSCs and cell lines correlates best with expression of another isoform, ALDH1A3, not ALDH1A1. We performed shRNA knockdown experiments of the various ALDH isoforms and found that only ALDH1A3 knockdown uniformly reduced ALDH activity of breast cancer cells. Immunohistological studies with fixed patient breast tumor samples revealed that ALDH1A3 expression in patient breast tumors correlates significantly with tumor grade, metastasis, and cancer stage. Our results, therefore, identify ALDH1A3 as a novel CSC marker with potential clinical prognostic applicability, and demonstrate a clear correlation between CSC prevalence and the development of metastatic breast cancer.

Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells.

Curcumin from the rhizome of theCurcuma longa plant has chemopreventative activity and inhibits the growth of neoplastic cells. Since p53 has been suggested to be important for anticancer activity by curcumin, we investigated curcumin-induced cytotoxicity in cultures of p53(+/+) and p53(-/-) HCT-116 colon cancer cells, as well as mutant p53 HT-29 colon cancer cells. Curcumin killed wild-type p53 HCT-116 cells and mutant p53 HT-29 cells in a dose- and time-dependent manner. In addition, curcumin-treated p53(+/+) HCT-116 cells and mutant p53 HT-29 cells showed upregulation of total and activated p53, as well as increased expression of p53-regulated p21, PUMA (p53 upregulated modulator of apoptosis), and Bax; however, an equivalent cytotoxic effect by curcumin was observed in p53(+/+) and p53(-/-) HCT-116 cells, demonstrating that curcumin-induced cytotoxicity was independent of p53 status. Similar results were obtained when the cytotoxic effect of curcumin was assessed in wild-type p53 HCT-116 cells after siRNA-mediated p53 knockdown. Chromatin condensation, poly (ADP-ribose) polymerase-1 cleavage and reduced pro-caspase-3 levels in curcumin-treated p53(+/+) and p53(-/-) HCT-116 cells suggested that curcumin caused apoptosis. In addition, exposure to curcumin resulted in superoxide anion production and phosphorylation of oxidative stress proteins in p53(+/+) and p53(-/-) HCT-116 cells. Collectively, our results indicate that, despite p53 upregulation and activation, curcumin-induced apoptosis in colon cancer cells was independent of p53 status and involved oxidative stress. Curcumin may therefore have therapeutic potential in the management of colon cancer, especially in tumorsthatare resistant to conventional chemotherapydue todefects inp53 expression or function.

Curcumin-induced apoptosis in PC3 prostate carcinoma cells is caspase-independent and involves cellular ceramide accumulation and damage to mitochondria.

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 microM curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and survivin expression and Akt signaling.

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long-term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin-induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time- and dose-dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild-type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53-independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP-ribose) polymerase-1 cleavage in curcumin-treated HEY cells, indicated cell death by apoptosis. Procaspase-3, procaspase-8, and procaspase-9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl-2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin-treated HEY cells. Curcumin also activated p38 mitogen-activated protein kinase (MAPK) without altering extracellular signal-regulated kinase 1/2 activity. We conclude that p53-independent curcumin-induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl-2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer.

Oncolytic reovirus effectively targets breast cancer stem cells.

Recent evidence suggests that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Hence, novel cancer therapies will need to be tested for both tumor regression and CSC targeting. Herein we show that oncolytic reovirus that induces regression of human breast cancer primary tumor samples xenografted in immunocompromised mice also effectively targets and kills CSCs in these tumors. CSCs were identified based on CD24(-)CD44(+) cell surface expression and overexpression of aldehyde dehydrogenase. Upon reovirus treatment, the CSC population was reduced at the same rate as non-CSCs within the tumor. Immunofluorescence of breast tumor tissue samples from the reovirus- and mock-treated mice confirmed that both CSCs and non-CSCs were infectible by reovirus, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay showed that both populations died by apoptosis. Ras, which has been shown to mediate reovirus oncolysis, was found to be present at similar levels in all cell types, and this is consistent with their comparable sensitivity to reovirus. These experiments indicate that oncolytic reovirus has the potential to induce tumor regression in breast cancer patients. More important, the CSC population was equally reduced and was as susceptible to reovirus treatment as the non-CSC population.

2-Chloro-2'-deoxyadenosine-induced apoptosis in T leukemia cells is mediated via a caspase-3-dependent mitochondrial feedback amplification loop.

2-Chloro-2'-deoxyadenosine (CdA; cladribine) is a chemotherapeutic agent used in the treatment of certain leukemias. However, the signalling events that govern CdA-mediated cytotoxicity in leukemia cells remain unclear. We show here that CdA treatment caused Jurkat human T leukemia cells to die via apoptosis in a dose- and time-dependent fashion. Bcl-2 overexpression protected Jurkat T leukemia cells from CdA-induced apoptosis and loss of mitochondrial transmembrane potential (Delta Psi m). Furthermore, mitochondria that were isolated from Jurkat T leukemia cells and then exposed to CdA showed a loss of Delta Psi m, indicating that CdA directly compromised outer mitochondrial membrane integrity. CdA treatment of Jurkat T leukemia cells resulted in the activation of caspase-3, -8, and -9, while inhibition of these caspases prevented the CdA-induced loss of Delta Psi m, as well as DNA fragmentation. In addition, caspase-3 inhibition prevented caspase-8 activation while caspase-8 inhibition prevented caspase-9 activation. Death receptor signalling was not involved in CdA-induced apoptosis since cytotoxicity was not affected by FADD-deficiency or antibody neutralization of either Fas ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Taken together, these data suggested that CdA-induced apoptosis in Jurkat T leukemia cells was mediated via a caspase-3-dependent mitochondrial feedback amplification loop. CdA treatment also increased p38 mitogen-activated protein (MAPK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in Jurkat T leukemia cells. Although ERK1/2 inhibition did not affect CdA-mediated cytotoxicity, inhibition of p38 MAPK had an enhancing effect, which suggested a cytoprotective function for p38 MAPK. Agents that inhibit p38 MAPK might therefore increase the effectiveness of CdA-based chemotherapy.