PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3.

BACKGROUND: The DDX3 helicase inhibitor RK-33 is a newly developed anticancer agent that showed promising results in preclinical research (Bol et al. EMBO Mol Med, 7(5):648-649, 2015). However, due to the physicochemical and pharmacological characteristics of RK-33, we initiated development of alternative formulations of RK-33 by preparing sustained release nanoparticles that can be administered intravenously. METHODS: In this study, RK-33 was encapsulated in poly(lactic-co-glycolic acid) (PLGA), one of the most well-developed biodegradable polymers, using the emulsion solvent evaporation method. RESULTS: Hydrodynamic diameter of RK-33-PLGA nanoparticles was about 245 nm with a negative charge, and RK-33-PLGA nanoparticles had a payload of 1.4 % RK-33. RK-33 was released from the PLGA nanoparticles over 7 days (90 ± 5.7 % released by day 7) and exhibited cytotoxicity to human breast carcinoma MCF-7 cells in a time-dependent manner. Moreover, RK-33-PLGA nanoparticles were well tolerated, and systemic retention of RK-33 was markedly improved in normal mice. CONCLUSIONS: PLGA nanoparticles have a potential as a parenteral formulation of RK-33.

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-ß-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

A 5' distal palindrome within the mouse mammary tumor virus-long terminal repeat recruits a mammary gland-specific complex and is required for a synergistic response to progesterone plus prolactin.

Progesterone (P) and prolactin (PRL) fulfill crucial roles during growth and differentiation of the mammary epithelium, and each has been implicated in the pathogenesis of mammary cancer. We previously identified that these hormones synergistically stimulate the proliferation of mouse mammary epithelial cells in vivo, although the mechanism(s) underlying their cooperative effect are unknown. We now report a novel pathway by which P and PRL synergize to activate transcription from the long terminal repeat (LTR) of the mouse mammary tumor virus-LTR (MMTV-LTR) in T47D breast cancer cells. Using serial 5' and 3' deletions of the MMTV-LTR, in addition to selective mutations, we identified that a previously uncharacterized inverted palindrome on the distal enhancer (-941/-930), in addition to a signal transducer and activator of transcription 5 site, was essential for the synergistic activation of transcription by P and PRL. Notably, hormone synergy occurred via a mechanism that was independent of the P receptor DNA-binding elements found in the proximal MMTV-LTR hormone-response element. The palindrome specifically recruited a protein complex (herein termed mammary gland-specific complex) that was almost exclusive to normal and cancerous mammary cells. The synergy between P and PRL occurred via a Janus kinase 2 and c-Src/Fyn-dependent signaling cascade downstream of P and PRL receptors. Combined, our data outline a novel pathway in T47D cells that may facilitate the action(s) of P and PRL during mammary development and breast cancer.

Ductal access for prevention and therapy of mammary tumors.

In cancer patients and in those at high risk, systemic exposure to agents for therapy or prevention is accompanied by undesirable side effects. We hypothesized that it is possible to prevent and treat breast cancer by introducing anticancer agents into the mammary ductal network. Here, we show the efficacy of intraductally administered anticancer agents 4-hydroxytamoxifen and pegylated liposomal doxorubicin (PLD) in the prevention and treatment of breast cancer using the rat N-methyl-N'-nitrosourea-induced and spontaneous HER-2/neu transgenic mouse (neu-N) models of breast cancer. Intraductal administration of PLD to neu-N mice caused regression of established tumors and prevented tumor development more effectively than i.v. injection (P < 0.0001). Intraductal administration resulted in lower circulating levels of PLD compared with i.v. administration, with no evidence of systemic toxicity or long-term histopathologic changes in the mammary gland. Compared with systemic administration, intraductal injection provides direct access to breast lesions with higher local and lower systemic drug exposure. These studies suggest that this approach has potential for application to prevention and neoadjuvant therapy of early breast cancer.

Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

Loss of the tight junction protein claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast.

Claudins are transmembrane proteins that seal tight junctions, and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Here, we report that Claudin-7 (CLDN-7) expression is lower in invasive ductal carcinomas (IDC) of the breast than in normal breast epithelium, as determined by both RT-PCR (9/10) and Western analysis (6/8). Immunohistochemical (IHC) analysis of ductal carcinoma in situ (DCIS) and IDC showed that the loss of CLDN-7 expression correlated with histological grade in both DCIS (P<0.001, n=38) and IDC (P=0.014, n=31), occurring predominantly in high-grade (Nuclear and Elston grade 3) lesions. Tissue array analysis of 355 IDC cases further confirmed the inverse correlation between CLDN-7 expression and histological grade (P=0.03). This pattern of expression is consistent with the biological function of CLDN-7, as greater discohesion is typically observed in high-grade lesions. In line with this observation, by IHC analysis, CLDN-7 expression was lost in the vast majority (13/17) of cases of lobular carcinoma in situ, which is defined by cellular discohesion. In fact, inducing disassociation of MCF-7 and T47D cells in culture by treating with HGF/scatter factor resulted in a loss of CLDN-7 expression within 24 h. Silencing of CLDN-7 expression correlated with promoter hypermethylation as determined by methylation-specific PCR (MSP) and nucleotide sequencing in breast cancer cell lines (3/3), but not in IDCs (0/5). In summary, these studies provide insight into the potential role of CLDN-7 in the progression and ability of breast cancer cells to disseminate.

Cyclin E is a target of WT1 transcriptional repression.

WT1 was originally identified as a Wilms' tumor suppressor gene, but it may have oncogenic potential in leukemia and in some solid tumors. WT1 is a transcription factor that has been implicated in the regulation of target genes related to apoptosis, genitourinary differentiation, and cell cycle progression. Because induction of WT1 leads indirectly to increased p21 expression in osteosarcoma cells, we investigated the possibility that other genes involved in the G(1)/S phase transition might also be WT1 targets. Cyclin E plays a crucial role in the cell cycle by activating cyclin-dependent kinase 2, which phosphorylates Rb, leading to progression from G(1) into S phase. We identified several WT1 binding sites in the cyclin E promoter. We demonstrate that WT1 binds to these sites and that in transient transfection assays WT1 represses the cyclin E promoter. This activity is dependent on the presence of a binding site located downstream of the transcription start site. In intact cells, induction of WT1 expression down-regulates cyclin E protein levels. These results provide the first demonstration that WT1 can directly modulate the expression of a gene involved in cell cycle progression.