BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment: implications for breast cancer prevention with antioxidant therapies.

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.

Hereditary ovarian cancer and two-compartment tumor metabolism: epithelial loss of BRCA1 induces hydrogen peroxide production, driving oxidative stress and NFκB activation in the tumor stroma.

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary ovarian cancers. Here, we used a co-culture approach to study the metabolic effects of BRCA1-null ovarian cancer cells on adjacent tumor-associated stromal fibroblasts. Our results directly show that BRCA1-null ovarian cancer cells produce large amounts of hydrogen peroxide, which can be abolished either by administration of simple antioxidants (N-acetyl-cysteine; NAC) or by replacement of the BRCA1 gene. Thus, the BRCA1 gene normally suppresses tumor growth by functioning as an antioxidant. Importantly, hydrogen peroxide produced by BRCA1-null ovarian cancer cells induces oxidative stress and catabolic processes in adjacent stromal fibroblasts, such as autophagy, mitophagy and glycolysis, via stromal NFκB activation. Catabolism in stromal fibroblasts was also accompanied by the upregulation of MCT4 and a loss of Cav-1 expression, which are established markers of a lethal tumor microenvironment. In summary, loss of the BRCA1 tumor suppressor gene induces hydrogen peroxide production, which then leads to metabolic reprogramming of the tumor stroma, driving stromal-epithelial metabolic coupling. Our results suggest that new cancer prevention trials with antioxidants are clearly warranted in patients that harbor hereditary/familial BRCA1 mutations.

Metabolic reprogramming of cancer-associated fibroblasts by TGF-ß drives tumor growth: connecting TGF-ß signaling with "Warburg-like" cancer metabolism and L-lactate production.

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-ß signaling. However, it remains unknown whether hyperactivation of the TGF-ß signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-ß ligands and the TGF-ß receptor I (TGFß-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-ß in tumorigenesis is compartment-specific, and that TGF-ß promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-ß are independent of the cell type generating TGF-ß. Thus, stromal-derived TGF-ß activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-ß-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-ß pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-ß ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-ß pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-ß pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-ß signaling and the metabolic reprogramming of the tumor microenvironment.

Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production.

Senescent fibroblasts are known to promote tumor growth. However, the exact mechanism remains largely unknown. An important clue comes from recent studies linking autophagy with the onset of senescence. Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST). To test this hypothesis, human fibroblasts immortalized with telomerase (hTERT-BJ1) were stably transfected with autophagy genes (BNIP3, CTSB or ATG16L1). Their overexpression was sufficient to induce a constitutive autophagic phenotype, with features of mitophagy, mitochondrial dysfunction and a shift toward aerobic glycolysis, resulting in L-lactate and ketone body production. Autophagic fibroblasts also showed features of senescence, with increased p21(WAF1/CIP1), a CDK inhibitor, cellular hypertrophy and increased ß-galactosidase activity. Thus, we genetically validated the existence of the autophagy-senescence transition. Importantly, autophagic-senescent fibroblasts promoted tumor growth and metastasis, when co-injected with human breast cancer cells, independently of angiogenesis. Autophagic-senescent fibroblasts stimulated mitochondrial metabolism in adjacent cancer cells, when the two cell types were co-cultured, as visualized by MitoTracker staining. In particular, autophagic ATG16L1 fibroblasts, which produced large amounts of ketone bodies (3-hydroxy-butyrate), had the strongest effects and promoted metastasis by up to 11-fold. Conversely, expression of ATG16L1 in epithelial cancer cells inhibited tumor growth, indicating that the effects of autophagy are compartment-specific. Thus, autophagic-senescent fibroblasts metabolically promote tumor growth and metastasis, by paracrine production of high-energy mitochondrial fuels. Our current studies provide genetic support for the importance of "two-compartment tumor metabolism" in driving tumor growth and metastasis via a simple energy transfer mechanism. Finally, ß-galactosidase, a known lysosomal enzyme and biomarker of senescence, was localized to the tumor stroma in human breast cancer tissues, providing in vivo support for our hypothesis. Bioinformatic analysis of genome-wide transcriptional profiles from tumor stroma, isolated from human breast cancers, also validated the onset of an autophagy-senescence transition. Taken together, these studies establish a new functional link between host aging, autophagy, the tumor microenvironment and cancer metabolism.

CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth.

Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-ß signaling, with increased transcription of TGF-ß target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-ß / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a stromal marker of poor clinical outcome in women with primary breast cancer, dissecting the downstream signaling effects of Cav-1 are important for understanding disease pathogenesis, and identifying novel therapeutic targets.

Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection.

Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as "fuel" for oxidative mitochondrial metabolism.  Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach.  TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis.  Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis, and treatment failure, resulting in poor clinical outcome in breast cancer patients. Thus, this new experimental model system, employing glycolytic fibroblasts, may be highly clinically relevant. These studies also have implications for understanding the role of hydrogen peroxide production in oxidative damage and "host cell aging," in providing a permissive metabolic microenvironment for promoting and sustaining tumor growth.

Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.

We have recently proposed a new model for understanding tumor metabolism, termed: "The Autophagic Tumor Stroma Model of Cancer Metabolism". In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ~4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified > 40 protein biomarkers that were upregulated, most of which were associated with i) myofibroblast differentiation, or ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant over-expression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with "The Autophagic Tumor Stroma Model of Cancer". The human Cav-1 deficient fibroblasts that we have generated are a new genetically tractable model system for identifying other suppressors of the cancer-associated fibroblast phenotype, via a genetic "complementation" approach. This has important implications for understanding the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER+ breast cancers, which are all associated with a Cav-1 deficient "lethal" tumor micro-environment, driving poor clinical outcome.

The autophagic tumor stroma model of cancer or "battery-operated tumor growth": A simple solution to the autophagy paradox.

The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the "Autophagy Paradox". We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm "The Autophagic Tumor Stroma Model of Cancer Cell Metabolism" or "Battery-Operated Tumor Growth". In this sense, autophagy in the tumor stroma serves as a "battery" to fuel tumor growth, progression and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients-both effectively "starving" cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy, by the upregulation of natural endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy, by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a "lethal" aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy, and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.

Sex differences in UDP-glucuronosyltransferase 2B17 expression and activity.

UDP-glucuronosyltransferases (UGTs) are enzymes involved in the metabolism of steroid hormones, carcinogens, cancer chemotherapy agents, and addictive agents from cigarettes. Because the UGT2B family of genes has been linked to hormonal regulation in human cell lines in vitro, we hypothesized that there may be sex-related differences in the expression and activity of these genes in human tissues. To evaluate whether there are sex differences in UGT2B expression and activity, we examined 103 normal human liver specimens for UGT2B expression by real-time polymerase chain reaction and in vitro glucuronidation activities in human liver microsomes (HLM). Men exhibited an approximately 4-fold higher level of expression of UGT2B17 than women (p = 0.007). Consistent with the increased expression of UGT2B17 in men, HLM from men also had a higher level of glucuronidation activity than HLM from women against three UGT2B17 substrates: 3-fold higher for 17-dihydroexemestane (p = 0.002); 3-fold higher for 3-hydroxycotinine (p < 0.001); and 1.5-fold higher for suberoylanilide hydroxamic acid (p = 0.014). When we stratified by UGT2B17 gene deletion genotype, similar patterns were observed for all three substrates, with HLM from men with the UGT2B17 (+/+) or (+/0) genotypes exhibiting significantly higher levels of glucuronidation activity against all three substrates compared with HLM from women. These data suggest that men have a higher amount of UGT2B17 glucuronidation activity then women. This sex difference in UGT2B17 gene expression and corresponding protein activity could potentially result in different levels of carcinogen detoxification or drug elimination in men versus women.

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells.

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a "lethal tumor micro-environment." Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a "metabolic" and "mutagenic" motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the "Reverse Warburg Effect"). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use "oxidative stress" in adjacent fibroblasts (i) as an "engine" to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the "field effect" in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively "contagious"--spread from cell-to-cell like a virus--creating an "oncogenic/mutagenic" field promoting widespread DNA damage.

Understanding the "lethal" drivers of tumor-stroma co-evolution: emerging role(s) for hypoxia, oxidative stress and autophagy/mitophagy in the tumor micro-environment.

We have recently proposed a new model for understanding how tumors evolve. To achieve successful "Tumor-Stroma Co-Evolution", cancer cells induce oxidative stress in adjacent fibroblasts and possibly other stromal cells. Oxidative stress in the tumor stroma mimics the effects of hypoxia, under aerobic conditions, resulting in an excess production of reactive oxygen species (ROS). Excess stromal production of ROS drives the onset of an anti-oxidant defense in adjacent cancer cells, protecting them from apoptosis. Moreover, excess stromal ROS production has a "Bystander-Effect", leading to DNA damage and aneuploidy in adjacent cancer cells, both hallmarks of genomic instability. Finally, ROS-driven oxidative stress induces autophagy and mitophagy in the tumor micro-environment, leading to the stromal over-production of recycled nutrients (including energy-rich metabolites, such as ketones and L-lactate). These recycled nutrients or chemical building blocks then help drive mitochondrial biogenesis in cancer cells, thereby promoting the anabolic growth of cancer cells (via an energy imbalance). We also show that ketones and lactate help "fuel" tumor growth and cancer cell metastasis and can act as chemo-attractants for cancer cells. We have termed this new paradigm for accelerating tumor-stroma co-evolution, "The Autophagic Tumor Stroma Model of Cancer Cell Metabolism". Heterotypic signaling in cancer-associated fibroblasts activates the transcription factors HIF1alpha and NFκB, potentiating the onset of hypoxic and inflammatory response(s), which further upregulates the autophagic program in the stromal compartment. Via stromal autophagy, this hypoxic/inflammatory response may provide a new escape mechanism for cancer cells during anti-angiogenic therapy, further exacerbating tumor recurrence and metastasis.

Characterization of UGTs active against SAHA and association between SAHA glucuronidation activity phenotype with UGT genotype.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple other cancers. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To characterize the UGTs active against SAHA, homogenates from HEK293 cell lines overexpressing UGT wild-type or variant UGT were used. The hepatic UGTs 2B17 and 1A9 and the extrahepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by V(max)/K(M) (16+/-6.5, 7.1+/-2.2, 33+/-6.3, and 24+/-2.4 nL x min(-1) x microg UGT protein(-1), respectively), with UGT2B17 exhibiting the lowest K(M) (300 micromol/L) against SAHA of any UGT in vitro. Whereas the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P<0.005) decrease in glucuronidation activity against SAHA compared with wild-type UGT1A8, the UGT1A8p.Cys277Tyr variant exhibited no detectable glucuronidation activity; a similar lack of detectable glucuronidation activity was observed for the UGT1A10p.Gly139Lys variant. To analyze the effects of the UGT2B17 gene deletion variant (UGT2B17*2) on SAHA glucuronidation phenotype, human liver microsomes (HLM) were analyzed for glucuronidation activity against SAHA and compared with UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P<0.01) decrease in glucuronidation activity and a 75% (P<0.002) increase in K(M) compared with HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Overall, these results suggest that several UGTs play an important role in the metabolism of SAHA and that UGT2B17-null individuals could potentially exhibit altered SAHA clearance rates with differences in overall response.

Glucuronidation of active tamoxifen metabolites by the human UDP glucuronosyltransferases.

Tamoxifen (TAM) is an antiestrogen that has been widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism and elimination of TAM and its major active metabolites 4-hydroxytamoxifen (4-OH-TAM) and 4-OH-N-desmethyl-TAM (endoxifen; 4-hydroxy-N-desmethyl-tamoxifen) is via glucuronidation. Although limited studies have been performed characterizing the glucuronidation of 4-OH-TAM, no studies have been performed on endoxifen. In the present study, characterization of the glucuronidating activities of human UDP glucuronosyltransferases (UGTs) against isomers of 4-OH-TAM and endoxifen was performed. Using homogenates of individual UGT-overexpressing cell lines, UGTs 2B7 approximately 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by Vmax/K(M), with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest K(M) (3.7 microM). As determined by Vmax/K(M), UGT1A10 exhibited the highest overall O-glucuronidating activity against cis-4-OH-TAM, 10-fold higher than the next-most active UGTs 1A1 and 2B7, but with UGT1A7 exhibiting the lowest K(M). Although both N- and O-glucuronidation occurred for 4-OH-TAM in human liver microsomes, only O-glucuronidating activity was observed for endoxifen; no endoxifen-N-glucuronidation was observed for any UGT tested. UGTs 1A10 approximately 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by Vmax/K(M) for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest K(M) (39.9 microM). Similar to that observed for cis-4-OH-TAM, UGT1A10 also exhibited the highest activity for cis-endoxifen. These data suggest that several UGTs, including UGTs 1A10, 2B7, and 1A8 play an important role in the metabolism of 4-OH-TAM and endoxifen.