Androgen receptor function and targeted therapeutics across breast cancer subtypes.

Despite significant progress in breast cancer (BC) therapy, it is globally the most commonly diagnosed cancer and leads to the death of over 650,000 women annually. Androgen receptor (AR) is emerging as a potential new therapeutic target in BC. While the role of AR is well established in prostate cancer (PCa), its function in BC remains incompletely understood. Emerging data show that AR's role in BC is dependent on several factors including, but not limited to, disease subtype, tumour microenvironment, and levels of circulating oestrogens and androgens. While targeting AR in PCa is becoming increasingly effective, these advances have yet to make any significant impact on the care of BC patients. However, this approach is increasingly being evaluated in BC and it is clear that improvements in our understanding of AR's role in BC will increase the likelihood of success for AR-targeted therapies. This review summarizes our current understanding of the function of AR across BC subtypes. We highlight limitations in our current knowledge and demonstrate the importance of categorizing BC subtypes effectively, in relation to determining AR activity. Further, we describe the current state of the art regarding AR-targeted approaches for BC as monotherapy or in combination with radiotherapy.

Protein Synthesis Inhibition Activity of Mesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations of Oncogenic Signaling Molecules and Secreted Growth Factors.

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24⁻48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.

The impact of mechanically stimulated muscle-derived stromal cells on aged skeletal muscle.

Perivascular stromal cells, including mesenchymal stem/stromal cells (MSCs), secrete paracrine factor in response to exercise training that can facilitate improvements in muscle remodeling. This study was designed to test the capacity for muscle-resident MSCs (mMSCs) isolated from young mice to release regenerative proteins in response to mechanical strain in vitro, and subsequently determine the extent to which strain-stimulated mMSCs can enhance skeletal muscle and cognitive performance in a mouse model of uncomplicated aging. Protein arrays confirmed a robust increase in protein release at 24h following an acute bout of mechanical strain in vitro (10%, 1Hz, 5h) compared to non-strain controls. Aged (24month old), C57BL/6 mice were provided bilateral intramuscular injection of saline, non-strain control mMSCs, or mMSCs subjected to a single bout of mechanical strain in vitro (4×104). No significant changes were observed in muscle weight, myofiber size, maximal force, or satellite cell quantity at 1 or 4wks between groups. Peripheral perfusion was significantly increased in muscle at 4wks post-mMSC injection (p<0.05), yet no difference was noted between control and preconditioned mMSCs. Intramuscular injection of preconditioned mMSCs increased the number of new neurons and astrocytes in the dentate gyrus of the hippocampus compared to both control groups (p<0.05), with a trend toward an increase in water maze performance noted (p=0.07). Results from this study demonstrate that acute injection of exogenously stimulated muscle-resident stromal cells do not robustly impact aged muscle structure and function, yet increase the survival of new neurons in the hippocampus.

Mesothelin-targeted immunotoxin RG7787 has synergistic anti-tumor activity when combined with taxanes.

Recombinant immunotoxins (RITs) are antibody-based therapeutics that carry a toxin payload. The RG7787 RIT targets the cancer antigen mesothelin to deliver a recombinantly-engineered, reduced immunogenicity variant of Pseudomonas exotoxin A (PE) to the cytosol where it inhibits protein synthesis. Here we demonstrate that maximal doses of RG7787 temporarily halt growth of pancreatic cancer tumor xenografts, similar to the approved drugs gemcitabine and nab-paclitaxel, however, combination of the RIT with nab-paclitaxel produces durable complete regressions in most mice. Synergy between taxane and anti-MSLN RITs has been previously demonstrated in mouse models, but direct interaction of the combination in cell culture was not observed. Here, we show that this favorable interaction occurs in cell culture, is dependent on the dose and duration of RG7787 exposure, requires the catalytically active PE, and still occurs with RIT targeting a non-MSLN surface antigen. Unexpectedly, the combination does not increase RG7787-mediated protein synthesis inhibition nor perturb downstream apoptotic markers of RIT-mediated killing, but does augment levels of acetylated tubulin, a marker of taxane activity. Taken together, these data suggest that PE increases cell sensitivity to taxane-mediated killing by increasing taxane-mediated microtubule stability and priming cells for apoptosis by decreasing levels of the pro-survival factor Mcl-1.

Protection of the Furin Cleavage Site in Low-Toxicity Immunotoxins Based on Pseudomonas Exotoxin A.

Recombinant immunotoxins (RITs) are fusions of an Fv-based targeting moiety and a toxin. Pseudomonas exotoxin A (PE) has been used to make several immunotoxins that have been evaluated in clinical trials. Immunogenicity of the bacterial toxin and off-target toxicity have limited the efficacy of these immunotoxins. To address these issues, we have previously made RITs in which the Fv is connected to domain III (PE24) by a furin cleavage site (FCS), thereby removing unneeded sequences of domain II. However, the PE24 containing RITs do not contain the naturally occurring disulfide bond around the furin cleavage sequence, because it was removed when domain II was deleted. This could potentially allow PE24 containing immunotoxins to be cleaved and inactivated before internalization by cell surface furin or other proteases in the blood stream or tumor microenvironment. Here, we describe five new RITs in which a disulfide bond is engineered to protect the FCS. The most active of these, SS1-Fab-DS3-PE24, shows a longer serum half-life than an RIT without the disulfide bond and has the same anti-tumor activity, despite being less cytotoxic in vitro. These results have significance for the production of de-immunized, low toxicity, PE24-based immunotoxins with a longer serum half-life.