Neal Flomenberg

MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are negative regulators of gene expression. To determine whether miRNAs are associated with cytogenetic abnormalities and clinical features in acute myeloid leukemia (AML), we evaluated the miRNA expression of CD34(+) cells and 122 untreated adult AML cases using a microarray platform. After background subtraction and normalization using a set of housekeeping genes, data were analyzed using Significance Analysis of Microarrays. An independent set of 60 untreated AML patients was used to validate the outcome signatures using real-time polymerase chain reaction. We identified several miRNAs differentially expressed between CD34(+) normal cells and the AML samples. miRNA expression was also closely associated with selected cytogenetic and molecular abnormalities, such as t(11q23), isolated trisomy 8, and FLT3-ITD mutations. Furthermore, patients with high expression of miR-191 and miR-199a had significantly worse overall and event-free survival than AML patients with low expression (overall survival: miR-191, P = .03; and miR-199a, P = .001, Cox regression). In conclusion, miRNA expression in AML is closely associated with cytogenetics and FLT3-ITD mutations. A small subset of miRNAs is correlated with survival.

Rapid Mobilization of CD34+ Cells Following Administration of the CXCR4 Antagonist AMD3100 to Patients With Multiple Myeloma and Non-Hodgkin's Lymphoma

PURPOSE Interactions between the chemokine receptor CXCR4 and its ligand stromal derived factor-1 regulate hematopoietic stem-cell trafficking. AMD3100 is a CXCR4 antagonist that induces rapid mobilization of CD34+ cells in healthy volunteers. We performed a phase I study assessing the safety and clinical effects of AMD3100 in patients with multiple myeloma (MM) and non-Hodgkins lymphoma (NHL). PATIENTS AND METHODS Thirteen patients (MM, n=7; NHL, n=6) received AMD3100 at a dose of either 160 microg/kg (n=6) or 240 microg/kg (n=7). WBC and peripheral blood (PB) CD34+ cell counts were analyzed at 4 and 6 hours following injection. RESULTS AMD3100 caused a rapid and statistically significant increase in the total WBC and PB CD34+ counts at both 4 and 6 hours following a single injection. The absolute CD34+ cell count increased from a baseline of 2.6 +/- 0.7/microL (mean +/- SE) to 15.6 +/- 3.9/microL and 16.2 +/- 4.3/microL at 4 hours (P=.002) and 6 hours after injection (P =.003), respectively. The absolute CD34+ cell counts observed at 4 and 6 hours following AMD3100 were higher in the 240 microg/kg group (19.3 +/- 6.9/microL and 20.4 +/- 7.6/microL, respectively) compared with the 160 microg/kg group (11.3 +/- 2.7/microL and 11.3 +/- 2.5/microL, respectively). The drug was well tolerated and only grade 1 toxicities were encountered. CONCLUSION AMD3100 appears to be a safe and effective agent for the rapid mobilization of CD34+ cells in patients who have received prior chemotherapy. Further studies in combination with granulocyte colony-stimulating factor in patients with lymphoid malignancies are warranted.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Evidence for a stromal-epithelial "lactate shuttle" in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts.

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.

Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia. Identification of host-derived antidonor allocytotoxic T lymphocytes.

Clinical trials with bone marrow depleted of donor T lymphocytes indicate that both the incidence and severity of graft-versus-host disease (GVHD) in patients undergoing bone marrow transplantation (BMT) for treatment of leukemia are greatly reduced. However, there has been a concurrent increase in the incidence of graft rejection, particularly among recipients of HLA-nonidentical marrow grafts. In order to investigate the nature of graft failure, peripheral blood mononuclear cells (PBMC) present at the time of graft failure have been characterized by phenotypic and functional analyses in 5 recipients of HLA-nonidentical marrow grafts. Rejection of HLA-nonidentical marrow grafts was associated with the emergence of host-derived T lymphocytes in all 5 patients. In 3 of these patients, the cells could be tested directly for cell-mediated cytotoxicity. Antidonor cytotoxicity was detected in each of these 3 patients. In one patient the target specificity of the cytotoxic lymphocytes was identified as the donor class I HLA antigen, HLA-B7. None of the patient PBMC mediated cytotoxicity against the natural killer cell target K562.

Ketones and lactate increase cancer cell “stemness”, driving recurrence, metastasis and poor clinical outcome in breast cancer: Achieving personalized medicine via metabolo-genomics

Previously, we showed that high-energy metabolites (lactate and ketones) “fuel” tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with “stemness,” including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced “gene signatures” were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the “cancer stem cell” phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific “classical” gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and “mitochondrial poison” metformin. Finally, we propose that this new approach to personalized cancer medicine be termed “Metabolo-Genomics,” which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.

Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the "reverse Warburg effect": a transcriptional informatics analysis with validation.

Cav-1 (-/-) deficient stromal cells are a new genetic model for myofibroblasts and cancer-associated fibroblasts. Using an unbiased informatics analysis of the transcriptional profile of Cav-1 (-/-) deficient mesenchymal stromal cells, we have now identified many of the major signaling pathways that are activated by a loss of Cav-1, under conditions of metabolic restriction (with low glucose media). Our informatics analysis suggests that a loss of Cav-1 induces oxidative stress, which mimics a constitutive pseudo-hypoxic state, leading to 1) aerobic glycolysis and 2) inflammation in the tumor stromal microenvironment. This occurs via the activation of 2 major transcription factors, namely HIF (aerobic glycolysis) and NF-kB (inflammation) in Cav-1 (-/-) stromal fibroblastic cells. Experimentally, we show that Cav-1 deficient stromal cells may possess defective mitochondria, due to the over-production of nitric oxide (NO), resulting in the tyrosine nitration of the mitochondrial respiratory chain components (such as complex I). Elevated levels of nitro-tyrosine were observed both in Cav-1 (-/-) stromal cells, and via acute knock-down with siRNA targeting Cav-1. Finally, metabolic restriction with mitochondrial (complex I) and glycolysis inhibitors was synthetically lethal with a Cav-1 (-/-) deficiency in mice. As such, Cav-1 deficient mice show a dramatically reduced mitochondrial reserve capacity. Thus, a mitochondrial defect in Cav-1 deficient stromal cells could drive oxidative stress, leading to aerobic glycolysis, and inflammation, in the tumor microenvironment. These stromal alterations may underlie the molecular basis of the “Reverse Warburg Effect”, and could provide the key to targeted anti-cancer therapies using metabolic inhibitors. In direct support of these findings, the transcriptional profile of Cav-1 (-/-) stromal cells overlaps significantly with Alzheimer’s disease, which is characterized by oxidative stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and mitochondrial dysfunction. We conclude that Cav-1 (-/-) deficient mice are a new whole-body animal model for an activated lethal tumor micro-environment, i.e., “tumor stroma” without the tumor. Since Cav-1 (-/-) mice are also an established animal model for pro-fibrotic disease, our current results may have implications for understanding the pathogenesis of scleroderma (systemic sclerosis) and pulmonary fibrosis, which are also related to abnormal mesenchymal stem cell function.

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating “synthetic” cancer associated fibroblasts (CAFs). More specifically, these “synthetic” CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors.

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.

Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: Visualizing the therapeutic effects of metformin in tumor tissue

We have recently proposed a new mechanism for explaining energy transfer in cancer metabolism. In this scenario, cancer cells behave as metabolic parasites, by extracting nutrients from normal host cells, such as fibroblasts, via the secretion of hydrogen peroxide as the initial trigger. Oxidative stress in the tumor microenvironment then leads to autophagy-driven catabolism, mitochondrial dys-function, and aerobic glycolysis. This, in turn, produces high-energy nutrients (such as L-lactate, ketones, and glutamine) that drive the anabolic growth of tumor cells, via oxidative mitochondrial metabolism. A logical prediction of this new “parasitic” cancer model is that tumor-associated fibroblasts should show evidence of mitochondrial dys-function (mitophagy and aerobic glycolysis). In contrast, epithelial cancer cells should increase their oxidative mitochondrial capacity. To further test this hypothesis, here we subjected frozen sections from human breast tumors to a staining procedure that only detects functional mitochondria. This method detects the in situ enzymatic activity of cytochrome C oxidase (COX), also known as Complex IV. Remarkably, cancer cells show an over-abundance of COX activity, while adjacent stromal cells remain essentially negative. Adjacent normal ductal epithelial cells also show little or no COX activity, relative to epithelial cancer cells. Thus, oxidative mitochondrial activity is selectively amplified in cancer cells. Although COX activity staining has never been applied to cancer tissues, it could now be used routinely to distinguish cancer cells from normal cells, and to establish negative margins during cancer surgery. Similar results were obtained with NADH activity staining, which measures Complex I activity, and succinate dehydrogenase (SDH) activity staining, which measures Complex II activity. COX and NADH activities were blocked by electron transport inhibitors, such as Metformin. This has mechanistic and clinical implications for using Metformin as an anti-cancer drug, both for cancer therapy and chemo-prevention. We also immuno-stained human breast cancers for a series of well-established protein biomarkers of metabolism. More specifically, we now show that cancer-associated fibroblasts over-express markers of autophagy (cathepsin B), mitophagy (BNIP3L), and aerobic glycolysis (MCT4). Conversely, epithelial cancer cells show the over-expression of a mitochondrial membrane marker (TOMM20), as well as key components of Complex IV (MT-CO1) and Complex II (SDH-B). We also validated our observations using a bioinformatics approach with data from >2,000 breast cancer patients, which showed the transcriptional upregulation of mitochondrial oxidative phosphorylation (OXPHOS) in human breast tumors (p < 10-20), and a specific association with metastasis. Therefore, upregulation of OXPHOS in epithelial tumor cells is a common feature of human breast cancers. In summary, our data provide the first functional in vivo evidence that epithelial cancer cells perform enhanced mitochondrial oxidative phosphorylation, allowing them to produce high amounts of ATP. Thus, we believe that mitochondria are both the “powerhouse” and “Achilles’ heel” of cancer cells.