Novel C-terminal heat shock protein 90 inhibitors target breast cancer stem cells and block migration, self-renewal, and epithelial–mesenchymal transition

In patients with triple-negative breast cancer (TNBC), evidence suggests that tumor-initiating cells (TIC) have stem cell-like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C-terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA-MB-468LN, MDA-MB-231) treated with the HSP90 inhibitors KU711, KU758, and 17-AAG showed a 50–80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self-renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose-dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C-terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self-renewal

Subcutaneous Delivery of Nanoconjugated Doxorubicin and Cisplatin for Locally Advanced Breast Cancer Demonstrates Improved Efficacy and Decreased Toxicity at Lower Doses Than Standard Systemic Combination Therapy In Vivo

Background—Combination cytotoxic agents in breast cancer carry dose-limiting toxicities. We hypothesize that nanocarrier-conjugated doxorubicin and cisplatin will have improved tumor efficacy with decreased systemic toxicity over standard drugs, even at lower doses. Methods—Female Nu/Nu mice were injected in the breast with human MDA-MB-468LN cells and treated with either standard or nanocarrier-conjugated combination therapy (doxorubicin +cisplatin) at 50% or 75% MTD, and monitored for efficacy and toxicity over 12-weeks. Results—Efficacy results for mice treated with HA-conjugated doxorubicin/cisplatin at 50% MTD include:[complete responses(CR)=5, partial responses(PR)=2, and stable disease(SD)=1]and for HA-conjugated dox/cis at 75% MTD:[CR=7,PR=1; all CR’s confirmed histologically]. In comparison, mice given standard dox/cis(50% MTD)demonstrated:[progressive disease(PD)=6, SD=1, and PR=1] and for standard dox/cis(75% MTD):[PD=5,SD=3; p<0.0001 on multivariate ANOVA]. At 75% MTD, standard drug-treated mice had significant weight loss compared to nanocarrier drug-treated mice(p<0.001). Conclusion—Subcutaneous nanocarrier-delivery of doxorubicin and cisplatin demonstrated significantly improved efficacy with decreased toxicity compared to standard agent combination therapy at all doses tested achieving complete pathologic tumor response

A practical and sensitive method of quantitating lymphangiogenesis in vivo

To address the inadequacy of current assays, we developed a directed in vivo lymphangiogenesis assay (DIVLA) by modifying an established directed in vivo angiogenesis assay. Silicon tubes (angioreactors) were implanted in the dorsal flanks of nude mice. Tubes contained either growth factor-reduced basement membrane extract (BME)-alone (negative control) or BME-containing vascular endothelial growth factor (VEGF)-D (positive control for lymphangiogenesis) or FGF-2/VEGF-A (positive control for angiogenesis) or a high VEGF-D-expressing breast cancer cell line MDA-MD-468LN (468-LN), or VEGF-D-silenced 468LN. Lymphangiogenesis was detected superficially with Evans Blue dye tracing and measured in the cellular contents of angioreactors by multiple approaches: lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) protein (immunofluorescence) and mRNA (qPCR) expression and a visual scoring of lymphatic vs blood capillaries with dual Lyve1 (or PROX-11 or Podoplanin)/Cd31 immunostaining in cryosections. Lymphangiogenesis was absent with BME, high with VEGF-D or VEGF-D-producing 468LN cells and low with VEGF-D-silenced 468LN. Angiogenesis was absent with BME, high with FGF-2/VEGF-A, moderate with 468LN or VEGF-D and low with VEGF-D-silenced 468LN. The method was reproduced in a syngeneic murine C3L5 tumor model in C3H/HeJ mice with dual Lyve1/Cd31 immunostaining. Thus, DIVLA presents a practical and sensitive assay of lymphangiogenesis, validated with multiple approaches and markers. It is highly suited to identifying pro-and anti-lymphangiogenic agents, as well as shared or distinct mechanisms regulating lymphangiogenesis vs angiogenesis, and is widely applicable to research in vascular/tumor biology. © 2013 USCAP, Inc. All rights reserved

Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN

Introduction and Objectives: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. Results: A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. Conclusion: Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype. © 2012 Majumder et al

A Novel Intralymphatic Nanocarrier-Delivery System for Cisplatin Therapy in Breast Cancer with Improved Tumor Efficacy and Lower Systemic Toxicity In Vivo

Background A lymphatically delivered nanoconjugate of cisplatin was evaluated in an orthotopic mouse model of locoregionally metastatic breast cancer (LABC) to determine if it can overcome some of the limitations of standard cisplatin therapy such as high systemic toxicity. Methods Human breast cancer cells (107 MDA-MB-468LN) were injected into the mammary fat pad of female nu/nu mice. Once tumor volume reached 50 mm3; intravenous cisplatin or subcutaneous hyaluronan-cisplatin [HA-cisplatin] nanoconjugate was given 1/week × 3 at 3.3 mg/kg (platinum basis). Results Nanoconjugates co-localized with the tumors after subcutaneous peritumoral injection and demonstrated improved efficacy to intravenous cisplatin. After one month, renal tubular hemorrhage and edema were more prevalent in the intravenous formulation compared to subcutaneous HA-cisplatin nanoconjugates. Conclusions This nanocarrier delivery platform focuses drug in the areas where tumor burden is greatest, potentially reducing systemic toxicity, and has future applicability as a neoadjuvant or adjuvant therapy for LABC

Development of lymphatic drug delivery platforms for the treatment of carcinomas

Many cancers, such as breast cancer, lung cancer, and head and neck cancer, preferentially metastasize via the lymphatic system prior to their systemic invasion. Conventional chemotherapy has limited penetration into the lymphatics due to its anatomy, because drug molecules have to diffuse from the blood capillaries into the surrounding tissues and further enter the lymph vessels to reach the diseased lymph nodes. Therefore, chemotherapy can be of limited benefit in treating lymph node disease. In addition, due to the systemic administration and wide distribution, conventional intravenous chemotherapy causes a variety of organ toxicities, including but not limited to: renal toxicity, cardiotoxicity, and hepatic toxicity. This limits its use in the clinic, expecially for the elderly or late-stage patients who cannot tolerate systemic toxicities. Therefore, there is a critical need to develop of a localized drug delivery platform that confines the anti-cancer drugs to the site of the disease. This dissertation focuses on the development of subcutaneously delivered drug platforms for the treatment of cancers that are known to be lymphatically metastatic. The goal is to improve the anti-cancer efficacy and reduce the non-specific organ toxicities of small molecule chemotherapeutic agents. To achieve this goal, three polymer-based anti-cancer conjugates: hyaluronan-cisplatin (HA-Pt), hyaluronan-doxorubicin (HA-DOX), and sugar star polymer-nitric oxide prodrug 1 (sugar-NO1), were synthesized, characterized, and evaluated in preclinical rodent models. The route of drug administration along with the controlled release of the drug from the platforms, both play an important role in improving the biopharmaceutical properties of these conjugates. This is demonstrated by the extended systemic rentention, as well as the elevated local accumulation of the drug in the draining lymph nodes of the tumor, when compared to standard intravenous chemotherapy. The HA-Pt and HA-DOX conjugates exhibited reduced renal toxicity and cardiotoxicity, respectively, relative to the conventional intravenous chemotherapy in rats. Because of the improved pharmarcokinetic and toxicological profiles of the polymer-drug conjugates, animals that were treated with the subcutaneous conjugates demonstrated enhanced inhibition of the tumor progression and increased survival rates. To understand the increased anti-cancer activity of the conjugates on a cellular level, the interaction and internalization of the nanocarriers into tumor cells was also investigated using a gold nanoparticle tagged HA-Pt conjugate and a fluorescently labeled HA-DOX conjugate. In the clinic, cisplatin and doxorubicin are often administered in combination with other anti-tumor agents as a "chemo cocktail" to prevent chemoresistance. Additionally, nitric oxide has been shown to improve the cytotoxicity of cisplatin therapy via a synergistic effect. Therefore, the development of a nitric oxide prodrug cisplatin combinational therapy, with increased anti-proliferative activity against cancer cells, was investigated; and it is also presented in this disssertation and compared to cisplatin treatment alone. In conclusion, this thesis work has led to the successful preclinical development of two localized drug delivery platforms and one promising candidate for the treatment of metastatic carcinomas. The motivation for drug delivery scientists to develop more efficacious and less toxic chemotherapies will be presented

Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR

Metastasis is respoMetastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors

Triazole Containing Novobiocin and Biphenyl Amides as Hsp90 C-Terminal Inhibitors

Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due to their ability to segregate client protein degradation from induction of the prosurvival heat shock response, which is a major detriment associated with Hsp90 N-terminal inhibitors under clinical investigation. Based upon prior SAR trends, a 1,2,3-triazole side chain was placed in lieu of the aryl side chain and attached to both the coumarin and biphenyl scaffold. Antiproliferative studies against SKBr3 and MCF-7 breast cancer cell lines demonstrated these triazole-containing compounds to exhibit improved activity. These compounds were shown to manifest Hsp90 inhibitory activity through Western blot analysis and represent a new scaffold upon which more potent inhibitors can be pursued

Synthesis and Biological Evaluation of Novobiocin Core Analogues as Hsp90 Inhibitors

Development of heat shock protein 90 (Hsp90) C‐terminal inhibitors has emerged as an exciting strategy for the treatment of cancer. Previous efforts have focused on modifications to the natural products novobiocin and coumermycin. Moreover, variations in both the sugar and amide moieties have been extensively studied, whereas replacements for the coumarin core have received less attention. Herein, 24 cores were synthesized with varying distances and angles between the sugar and amide moieties. Compounds that exhibited good anti‐proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity, were those that placed the sugar and amide moieties between 7.7 and 12.1 Å apart along with angles of 180°.Angle of attack: The distance and angle between the N‐methylpiperidine and the biaryl side chain was analyzed in an effort to develop more potent Hsp90 C‐terminal inhibitors. Compounds that exhibited good anti‐proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity were those that placed the sugar and amide moieties between 7.7 and 12.1 Å apart along with angles of 180°.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136281/1/chem201504955_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136281/2/chem201504955.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136281/3/chem201504955-sup-0001-misc_information.pd

Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

INTRODUCTION AND OBJECTIVES: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. RESULTS: A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. CONCLUSION: Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype