Sudipa Saha Roy

Role of Estrogen Receptor Signaling in Breast Cancer Metastasis

Metastatic breast cancer is a life-threatening stage of cancer and is the leading cause of death in advanced breast cancer patients. Estrogen signaling and the estrogen receptor (ER) are implicated in breast cancer progression, and the majority of the human breast cancers start out as estrogen dependent. Accumulating evidence suggests that ER signaling is complex, involving coregulatory proteins and extranuclear actions. ER-coregualtory proteins are tightly regulated under normal conditions with miss expression primarily reported in cancer. Deregulation of ER coregualtors or ER extranuclear signaling has potential to promote metastasis in ER-positive breast cancer cells. This review summarizes the emerging role of ER signaling in promoting metastasis of breast cancer cells, discusses the molecular mechanisms by which ER signaling contributes to metastasis, and explores possible therapeutic targets to block ER-driven metastasis.

Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Significance of PELP1 in ER-Negative Breast Cancer Metastasis

Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer. Mol Cancer Res; 10(1); 25–33. ©2011 AACR.

Therapeutic Targeting of PELP1 Prevents Ovarian Cancer Growth and Metastasis

Purpose: Ovarian cancer remains a major threat to womens health, partly due to difficulty in early diagnosis and development of metastases. A critical need exists to identify novel targets that curb the progression and metastasis of ovarian cancer. In this study, we examined whether the nuclear receptor coregulator PELP1 (proline-, glutamic acid-, leucine-rich protein-1) contributes to progression and metastatic potential of ovarian cancer cells and determined whether blocking of the PELP1 signaling axis had a therapeutic effect. Experimental Design: Ovarian cancer cells stably expressing PELP1-shRNA (short hairpin RNA) were established. Fluorescent microscopy, Boyden chamber, invasion assays, wound healing, and zymography assays were performed to examine the role of PELP1 in metastasis. Expression analysis of the model cells was conducted using tumor metastasis microarray to identify PELP1 Target genes. Therapeutic potential of PELP1-siRNA in vivo was determined using a nanoliposomal formulation of PELP1-siRNA-DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) administered systemically in a xenograft model. Results: PELP1 knockdown caused cytoskeletal defects and significantly affected the migratory potential of ovarian cancer cells. Microarray analysis revealed that PELP1 affected the expression of selective genes involved in metastasis including Myc, MTA1, MMP2, and MMP9. Zymography analysis confirmed that PELP1 knockdown caused a decrease in the activation of matrix metalloproteases (MMP) 2 and MMP9. Compared with control siRNA-DOPC–treated mice, animals injected with PELP1-siRNA-DOPC had 54% fewer metastatic tumor nodules, exhibited a 51% reduction in tumor growth and an 84% reduction in ascites volume. Conclusion: The results suggest that PELP1 signaling axis is a potential druggable target and liposomal PELP1-siRNA-DOPC could be used as a novel drug to prevent or treat ovarian metastasis. Clin Cancer Res; 17(8); 2250–9. ©2011 AACR.

Synthesis of a novel, sequentially active-targeted drug delivery nanoplatform for breast cancer therapy

Breast cancer is the leading cause of cancer deaths among women. Paclitaxel (PTX), an important breast cancer medicine, exhibits reduced bioavailability and therapeutic index due to high hydrophobicity and indiscriminate cytotoxicity. PTX encapsulation in one-level active targeting overcomes such barriers, but enhances toxicity to normal tissues with cancer-similar expression profiles. This research attempted to overcome this challenge by increasing selectivity of cancer cell targeting while maintaining an ability to overcome traditional pharmacological barriers. Thus, a multi-core, multi-targeting construct for tumor specific delivery of PTX was fabricated with (i) an inner-core prodrug targeting the cancer-overexpressed cathepsin B through a cathepsin B-cleavable tetrapeptide that conjugates PTX to a poly(amidoamine) dendrimer, and (ii) the encapsulation of this prodrug (PGD) in an outer core of a RES-evading, folate receptor (FR)-targeting liposome. Compared to traditional FR-targeting PTX liposomes, this sequentially active-targeted dendrosome demonstrated better prodrug retention, an increased cytotoxicity to cancer cells (latter being true when FR and cathepsin B activities were both at moderate-to-high levels) and higher tumor reduction. This research may eventually evolve a product platform with reduced systemic toxicity inherent with traditional chemotherapy and localized toxicity inherent to single-target nanoplatforms, thereby allowing for better tolerance of higher therapeutic load in advanced disease states.

Design of a Paclitaxel Prodrug Conjugate for Active Targeting of an Enzyme Upregulated in Breast Cancer Cells

Breast cancer is the second most common cause of cancer-related deaths in women. Chemotherapy is an important treatment modality, and paclitaxel (PTX) is often the first-line therapy for its metastatic form. The two most notable limitations related to PTX-based treatment are the poor hydrophilicity of the drug and the systemic toxicity due to the drugs nonspecific and indiscriminate distribution among the tissues. The present work describes an approach to counter both challenges by designing a conjugate of PTX with a hydrophilic macromolecule that is coupled through a biocleavable linker, thereby allowing for active targeting to an enzyme significantly upregulated in cancer cells. The resultant strategy would allow for the release of the active ingredient preferentially at the site of action in related cancer cells and spare normal tissue. Thus, PTX was conjugated to the hydrophilic poly(amdioamine) [PAMAM] dendrimer through the cathepsin B-cleavable tetrapeptide Gly-Phe-Leu-Gly. The PTX prodrug conjugate (PGD) was compared to unbound PTX through in vitro evaluations against breast cancer cells and normal kidney cells as well as through in vivo evaluations using xenograft mice models. As compared to PTX, PGD demonstrated a higher cytotoxicity specific to cell lines with moderate-to-high cathepsin B activity; cells with comparatively lower cathepsin B activity demonstrated an inverse of this relationship. Regression analysis between the magnitude of PGD-induced cytotoxic increase over PTX and cathepsin B expression showed a strong, statistically significant correlation (r(2) = 0.652, p < 0.05). The PGD conjugate also demonstrated a markedly higher tumor reduction as compared to PTX treatment alone in MDA-MB-231 tumor xenograft models, with PGD-treated tumor volumes being 48% and 34% smaller than PTX-treated volumes at weeks 2 and 3 after treatment initiation.

TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans

Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

BU-32: a novel proteasome inhibitor for breast cancer

IntroductionProteasome inhibition provides an attractive approach to cancer therapy and may have application in the treatment of breast cancer. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade®, also called PS-341) in metastatic breast cancer patients have shown limited activity when used as a single agent. This underscores the need to find new and more efficacious proteasome inhibitors. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499-S) using in vitro and in vivo breast cancer models.MethodsWe have recently synthesized a novel proteasome inhibitor (BU-32) and tested its growth inhibitory effects in different breast cancer cells including MCF-7, MDA-MB-231, and SKBR3 by in vitro cytotoxicity and proteasomal inhibition assays. The apoptotic potential of BU32 was tested using flow cytometry and analyzing cell cycle regulatory proteins. In vivo tumor xenograft studies for solid tumor as well as tumor metastasis were conducted using MDA-MB-231-GFP cells.ResultsWe report for the first time that BU-32 exhibits strong cytotoxicity in a panel of cell lines: MDA-MB-231 (IC50 = 5.8 nM), SKBR3 (IC50 = 5.7 nM) and MCF-7 cells (IC50 = 5.8 nM). It downregulates a wide array of angiogenic marker genes and upregulates apoptotic markers, including Bid and Bax. Incubation of MDA-MB-231 cells with BU-32 results in the accumulation of cell cycle inhibitor proteins p21 and p27 and stabilization of the tumor suppressor protein p53. Studies in in vivo solid tumor and metastasis models show significant effect with a 0.06 mg/kg dose of BU-32 and marked reduction in tumor burden in the skeleton.ConclusionsWe have shown that BU-32 is effective in cultured breast cancer cells and in breast cancer xenografts. The results suggest its potential benefit in breast cancer treatment.

Design, synthesis, and biological evaluation of bone-targeted proteasome inhibitors for multiple myeloma

Multiple myeloma (MM) is an incurable neoplasm characterized by devastating and progressive bone destruction. Standard chemotherapeutic agents have not been effective at significantly prolonging the survival of MM patients and these agents are typically associated with often severe, dose-limiting side effects. There is great need for methods to target the delivery of novel, effective cytotoxic agents specifically to bone, where myeloma cells reside. We have synthesized and evaluated the effects of the bone-targeted proteasome inhibitors PS-341-BP-1, PS-341-BP-2 and MG-262-BP on cell proliferation using the mouse 5TGM1 and human RPMI 8226 cell lines in vitro. The compounds exhibit strong cytotoxicity on MM cell lines and reduce the number of viable cells in a dose dependent manner.

Novel role of PELP1 in regulating chemotherapy response in mutant p53-expressing triple negative breast cancer cells.

Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.