Ranjana Mitra

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)–positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC50, 12-24 μmol/L) and ER-negative (IC50, 45 μmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 μmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 μmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

CYP3A4 Mediates Growth of Estrogen Receptor-positive Breast Cancer Cells in Part by Inducing Nuclear Translocation of Phospho-Stat3 through Biosynthesis of (±)-14,15-Epoxyeicosatrienoic Acid (EET)

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G2/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.

Prediction of Postoperative Recurrence-Free Survival in Non-small Cell Lung Cancer by Using an Internationally Validated Gene Expression Model

Purpose: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non–small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. Experimental Design: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. Results: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). Conclusion: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients. Clin Cancer Res; 17(9); 2934–46. ©2011 AACR.

The human immunodeficiency virus protease inhibitor ritonavir inhibits lung cancer cells, in part, by inhibition of survivin

Introduction: Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. Methods: RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. Results: Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 &mgr;M). Conclusions: Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Abstract 357: CYP3A4 Activates Stat3 in ER + breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CYP3A4 expression in breast cancer has been reported to correlate with nodal metastases and decreased overall survival, but its mechanisms in cancer progression are unknown. ERalpha has been reported to promote CYP3A4 expression. We have previously demonstrated that CYP3A4 is an arachidonic acid (AA) epoxygenase that activates breast cancer cell proliferation through biosynthesis of 14,15-epoxyeicosatrienoic acid (EET). Methods: Effects of 14,15-EET on Stat3 regulation were studied in MCF7 lines expressing CYP3A4 shRNA. Quantitative immunofluorescence (AQUA) interrogation of a tissue microarray was performed to evaluate CYP3A4 in cytoplasm, ERalpha and pY705Stat3 (pYStat3) in nuclei, and HER2 in cytoplasm/membrane. Values were transformed using the natural logarithm. These values were then averaged across the two spot sets. Pearsons correlation was calculated between the markers of interest. Results: Stat3 (Tyr 705) phosphorylation is inhibited by CYP3A4 silencing. CYP3A4 synthesizes 14,15-EET in breast cancer cells and silencing of Stat3 blocks breast cancer cell growth and abrogates 14,15-EET-induced proliferation, indicating that CYP3A4 requires 14,15-EET biosynthesis and Stat3 to induce cell growth. Silencing of CYP3A4 reduces nuclear pY705-Stat3, while 14,15-EET restores this signaling process. By preliminary immunofluorescence study of consecutive breast cancers (n=48) nuclear ERalpha was associated with cytoplasmic CYP3A4 with a correlation of r=0.7575 (p-value < 0.0001). Cytoplasmic CYP3A4 correlated with nuclear pYStat3, with a correlation of r=0.2708, approaching statistical significance (p = 0.0656). Cytoplasmic/membrane HER2, known to be induced by Stat3, was associated with nuclear pYStat3, exhibiting a correlation of r=0.4460 (p-value = 0.0015). Conclusions: ER+ breast cancer cell growth is therefore mediated, in part, by CYP3A4 biosynthesis of 14,15-EET that induces nuclear pY705-Stat3. These studies indicate that CYP3A4 is an AA epoxygenase that is expressed in ER+ breast cancer and promotes Stat3-mediated breast cancer cell growth, in part, through 14,15-EET biosynthesis. Activated Stat3 may mediate cytoplasmic/membrane HER2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 357. doi:10.1158/1538-7445.AM2011-357

Abstract 4444: Desthiazolylmethyloxycarbonyl ritonavir is a candidate proteasome activator drug in breast cancer that down-regulates survivin and HER2

Purpose: Desthiazolylmethyloxycarbonyl ritonavir (M1) is a minor metabolite of ritonavir, but exhibits more potent activity against ER+, HER2+ and triple negative breast cancer lines. The mechanisms of action of M1 are unknown and may be very different from ritonavir. This study investigates M1 effects on proteasome activity and unfolded protein response (UPR), which have been identified as ritonavir targets. Methods: Assays for cell growth, cell cycle progression and apoptosis were used to determine the mechanisms of M1 action in ER+ (T47D), HER2+ (SKBR3) and triple negative (MDA-MB-231) breast cancer lines. Effects of M1 on proteasome activity were determined by biochemical and cell-based luciferase assays. Effects of M1 on proteasome substrates and the UPR were determined by Western blotting and flow cytometry. Effects of M1 in vivo were determined using a xenograft model. Results: M1, compared to ritonavir, exhibited a significantly lower IC50 for the T47D, MDA-MB-231 and SKBR3 lines, but a higher IC50 for the non-transformed line MCF10A, indicating M1 selectivity for breast cancer. M1 induced a G0/G1 block and apoptosis in all lines tested. M1 at its IC50 significantly reduced survivin levels in the T47D and MDA-MB-231 lines, and surface HER2 in the SKBR3 line. However, it did not affect the expression of other short half-life antiapoptotic proteins, including MCL-1 and Bcl-2. In vivo, M1 inhibited the MDA-MB-231 xenograft at its maximum tolerated dose (MTD, 20 mg/kg), while ritonavir exhibited no effect at this dosing, which is half of its MTD. 1 h following M1 ip administration, M1 plasma levels were measurable (3.6 ± 2.3 microM). In contrast, ritonavir administration resulted in M1 levels that were at or below the limit of detection, indicating that M1 plasma levels associated with tumor response cannot be achieved with identical dosing of ritonavir. Remarkably, M1 up-regulated the chymotryptic, tryptic and caspase-like activities of the purified 20S proteasome, whereas ritonavir did not. M1 also up-regulated the proteasome chymotryptic site in breast cancer cells. Furthermore, M1 activation of the proteasome was abrogated by lactacystin or MG132. Sp3, a survivin transcription factor that is known to be degraded by the proteasome, was reduced in M1-treated MDA-MB-231 cells (P Conclusions: M1 is more potent than ritonavir in vitro and in vivo and, in contrast to ritonavir, is an activator of the proteasome, suggesting a novel mechanism of anticancer activity. Survivin may be an important target of M1 in ER+ and triple negative breast cancer, while HER2 is a target in HER2+ breast cancer. M1, a candidate HIV protease inhibitor derivative, is promising for clinical development in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4444.

Abstract 4011: CYP3A4 over-expression promotes ER-positive breast cancer cell growth, in part, through Stat3 activation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cytochrome P450 3A4 (CYP3A4) is an important monooxygenase that metabolizes a wide range of xenobitics including many drugs and environmental chemicals, as well as endogenous substances such as steroids. Expression of CYP3A4 has been observed in breast tumors, but its studies in breast cancer cells have mainly focused on carcinogen bioactivation, hormone metabolism and chemotherapy resistance. Whether CYP3A4 has a direct effect on breast cancer progression is unknown. To address these questions, we performed RNAi knock down studies, demonstrating roles for CYP3A4 in proliferation, motility, and clonogenicity of the MCF7 line. CYP3A4 knock down also inhibited phosphorylation of key regulatory proteins such as c-Src and STAT3, implicating its importance in breast cancer progression. To further study the effect and mechanisms of CYP3A4 in breast cancer progression, stable CYP3A4 over-expressing MCF7 cell lines were isolated and characterized. CYP3A4 over-expressing clones exhibited a higher proliferation rate than the vector control clone, consistent with the flow cytometry cell cycle analysis in which CYP3A4 over-expressing clones exhibited a markedly higher S phase component. Apoptosis was not increased due to CYP3A4 over-expression. The CYP3A4 over-expressing clones adhere poorly to plastic and require poly-D-lysine coated tissue culture plate to grow robustly. Adhesion to fibronectin was also reduced as the result of over-expression. In immunofluorescence microscopic study, the highest CYP3A4 over-expressing clone displayed disorganized focal adhesions, potentially explaining the poor adhesion. Consistent with the knock down study, CYP3A4 over-expressing clones exhibited elevated phosphorylation of both c-Src and Stat3 that could be responsible for the observed promotion of proliferation. These results support the model that CYP3A4 is an important signaling protein, linked to the c-Src/Stat3 pathway that influences breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4011.

Abstract 4129: Effects of the metabolism of omega-3 fatty acid EPA by CYP1A1 in breast cancer proliferation and survival

Omega-3 fatty acids are widely known for their importance in heart disease. However, the impact of omega-3 fatty acids in breast cancer is not fully understood. Through these studies we seek to understand the role of the metabolism of the omega-3 fatty acid known as eicosapentaenoic acid (EPA) in breast cancer and determine whether it may affect breast cancer progression. It is known that high levels (60uM) of EPA have anti-proliferative effects in various cancer lines. Consistent with these reports, we found that EPA treatment (60uM) of the estrogen receptor positive line MCF7 and the estrogen receptor negative line MDA-MB-231 decreases cell proliferation. Interestingly, lower dosing of EPA (15uM-40uM) significantly stimulated the proliferation of these lines. It has been shown that cytochrome P450 1A1 (CYP1A1) selectively epoxygenates EPA to 17(18)-epoxyeicosatetraenoic acid (17,18-EpETE). Measurement of cell growth by MTT assay shows that 17,18-EpETE promotes the proliferation of the MCF7 and MDA-MB-231 lines in a dose- (≥ 1uM) and time-dependent (max effect at 48-72hrs) manner. Together, these results led us to hypothesize that CYP1A1 metabolizes EPA into 17,18-EpETE, thereby promoting breast cancer proliferation and survival. To better understand this epoxygenase mechanism, we studied the impact of CYP1A1 and the 17,18-EpETE metabolite of EPA on cell proliferation by MTT assay, cell cycle progression by propidium iodine staining, and apoptosis by Annexin V flow cytometry. We found that CYP1A1 is expressed in MCF7 and MDA-MB-231 lines and siRNA-mediated knock down significantly inhibits the proliferation of these lines. CYP1A1 knock down also blocks the proliferative effect of treatment with 15uM EPA. Our preliminary data also indicate that 17,18-EpETE promotes growth of the MDA-MB-231 line, in part, by decreasing apoptosis and increasing the S- and G2/M cell populations. Consistently, knock down of CYP1A1 in MDA-MB-231 line increases the G0/1 cell population and decreases number of cells in the S and G2/M phases, consistent with a G1 block. The mechanism of action in MCF7 cells is yet to be determined. Better understanding of the mechanism by which EPA metabolism affects breast cancer may impact our understanding of breast cancer prevention and therapeutics. We propose that CYP1A1 may be of significant importance in breast cancer progression through novel mechanisms that depend on biosynthetic pathways promoting EPA epoxygenation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4129.