Targeting hedgehog-driven mechanisms of drug-resistant cancers.

Due to the cellular plasticity that is inherent to cancer, the acquisition of resistance to therapy remains one of the biggest obstacles to patient care. In many patients, the surviving cancer cell subpopulation goes on to proliferate or metastasize, often as the result of dramatically altered cell signaling and transcriptional pathways. A notable example is the Hedgehog (Hh) signaling pathway, which is a driver of several cancer subtypes and aberrantly activated in a wide range of malignancies in response to therapy. This review will summarize the field's current understanding of the many roles played by Hh signaling in drug resistance and will include topics such as non-canonical activation of Gli proteins, amplification of genes which promote tolerance to chemotherapy, the use of hedgehog-targeted drugs and tool compounds, and remaining gaps in our knowledge of the transcriptional mechanisms at play.

Bioinformatics Screen Reveals Gli-Mediated Hedgehog Signaling as an Associated Pathway to Poor Immune Infiltration of Dedifferentiated Liposarcoma.

Liposarcomas are the most diagnosed soft tissue sarcoma, with most cases consisting of well-differentiated (WDLPS) or dedifferentiated (DDLPS) histological subtypes. While both tumor subtypes can have clinical recurrence due to incomplete resections, DDLPS often has worse prognosis due to a higher likelihood of metastasis compared to its well-differentiated counterpart. Unfortunately, targeted therapeutic interventions have lagged in sarcoma oncology, making the need for molecular targeted therapies a promising future area of research for this family of malignancies. In this work, previously published data were analyzed to identify differential pathways that may contribute to the dedifferentiation process in liposarcoma. Interestingly, Gli-mediated Hedgehog signaling appeared to be enriched in dedifferentiated adipose progenitor cells and DDLPS tumors, and coincidentally Gli1 is often co-amplified with MDM2 and CDK4, given its genomic proximity along chromosome 12q13-12q15. However, we find that Gli2, but not Gli1, is differentially expressed between WDLPS and DDLPS, with a noticeable co-expression signature between Gli2 and genes involved in ECM remodeling. Additionally, Gli2 co-expression had a noticeable transcriptional signature that could suggest Gli-mediated Hedgehog signaling as an associated pathway contributing to poor immune infiltration in these tumors.

Nanoparticle STING Agonist Reprograms the Bone Marrow to an Antitumor Phenotype and Protects Against Bone Destruction.

When breast cancer metastasizes to bone, treatment options are limited. Failure to treat bone metastases is thought to be due to therapy-resistant features of the bone marrow microenvironment. Using a murine model of bone metastatic mammary carcinoma, we demonstrate that systemic delivery of polymer nanoparticles loaded with cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) inhibited tumor growth and bone destruction after 7 days of treatment. Each dose of STING-activating nanoparticles trafficked to the bone marrow compartment and was retained within the tumor microenvironment for over 24 hours, enhancing antitumor immunity through proinflammatory cytokine production and early T-cell activation. While acquired resistance mechanisms, including increased levels of immunosuppressive cytokines and the infiltration of regulatory T cells, ultimately limited antitumor efficacy after 2 weeks of treatment, bone protective effects remained. Overall, these studies demonstrate that STING pathway activation, here enabled using a nanomedicine approach to enhance CDN delivery to bone metastatic sites, can reprogram the immune contexture of the bone marrow to an antitumor phenotype that inhibits bone colonization of metastatic breast cancer cells and protects from tumor-mediated bone destruction. Significance: Bone metastases are difficult to treat due to the inaccessibility of the bone marrow compartment and the immunosuppressive microenvironment that protects resident stem cells. Packaging a STING agonist into a nanoparticle that enables systemic administration and drug accumulation at tumor sites overcomes both barriers to stymie metastatic breast cancer growth.

3D Bone Morphology Alters Gene Expression, Motility, and Drug Responses in Bone Metastatic Tumor Cells.

Patients with advanced skeletal metastases arising from primary cancers including breast, lung, and prostate suffer from extreme pain, bone loss, and frequent fractures. While the importance of interactions between bone and tumors is well-established, our understanding of complex cell-cell and cell-microenvironment interactions remains limited in part due to a lack of appropriate 3D bone models. To improve our understanding of the influence of bone morphometric properties on the regulation of tumor-induced bone disease (TIBD), we utilized bone-like 3D scaffolds in vitro and in vivo. Scaffolds were seeded with tumor cells, and changes in cell motility, proliferation, and gene expression were measured. Genes associated with TIBD significantly increased with increasing scaffold rigidity. Drug response differed when tumors were cultured in 3D compared to 2D. Inhibitors for Integrin ß3 and TGF-ß Receptor II significantly reduced bone-metastatic gene expression in 2D but not 3D, while treatment with the Gli antagonist GANT58 significantly reduced gene expression in both 2D and 3D. When tumor-seeded 3D scaffolds were implanted into mice, infiltration of myeloid progenitors changed in response to pore size and rigidity. This study demonstrates a versatile 3D model of bone used to study the influence of mechanical and morphometric properties of bone on TIBD.

Tuning Ligand Density To Optimize Pharmacokinetics of Targeted Nanoparticles for Dual Protection against Tumor-Induced Bone Destruction.

Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo. In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)-block-(alendronate acrylamide-co-N,N-dimethylacrylamide)] [PPS-b-P(Aln-co-DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Aln conferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations that drug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Pilot study of a high-frequency school-based hearing screen to detect adolescent hearing loss.

OBJECTIVE: Like most of the United States, school-based hearing screening in Pennsylvania focuses on low-frequency, conductive hearing losses typical for young children, rather than the high-frequency, noise-induced hearing loss more prevalent among adolescents. The objective of this study was to compare the sensitivity and specificity of current school hearing screening in Pennsylvania with hearing screening including high frequencies, designed to detect adolescent hearing loss. SETTING: A single public high school. METHODS: In the Autumn of 2011 the high-frequency screen was delivered alongside the Pennsylvania school screen for students in the 11(th) grade. Screening referrals and a subset of passes returned for "gold standard" testing with audiology in a sound treated booth, in order to determine the sensitivity and specificity of the screening tests. RESULTS: Of 282 participants, five (2%) were referred on the Pennsylvania school screen, and 85 (30%) were referred on the high-frequency screen. Of the 48 who returned for gold standard testing with audiology, hearing loss was diagnosed in 9/48 (19%). Sensitivity of the Pennsylvania and high-frequency screens were 13% (95% confidence interval [CI] 0-53%) and 100% (95% CI 66-100%) respectively. Specificity of the Pennsylvania and high-frequency screens were 97% (95% CI 87-100%) and 49% (95% CI 32-65%) respectively. CONCLUSIONS: Current school hearing screens have low sensitivity for detection of adolescent hearing loss. Modifying school-based protocols may be warranted to best screen adolescents, and make optimal use of school nurse time and effort.

Improving detection of adolescent hearing loss.

OBJECTIVES: To compare a protocol for pure-tone threshold testing, capable of detecting high-frequency hearing loss as indicated by notched audiometric configurations, with the current school rapid hearing screen and to determine typical adolescent noise exposures associated with notched audiometric configurations. DESIGN: In conjunction with required school rapid hearing screening, a pure-tone threshold testing protocol was administered, specifically to test hearing at high frequencies. A single audiologist reviewed the results. Students completed a survey assessing their noise exposures. SETTING: A public high school in Pennsylvania. PARTICIPANTS: Eleventh-grade students. MAIN OUTCOME MEASURE: Notched audiometric configurations on the pure-tone threshold test. RESULTS: Among 296 participants, 78 (26.4%) failed pure-tone threshold testing compared with 15 (5.1%) failing rapid hearing screening. Among those failing the pure-tone threshold testing, 67 (85.9%) failed due to notched audiometric configurations. Self-reported headphone use with an MP3 player was significantly associated with notched audiometric configurations compared with use of earbuds or stereo connection/docking systems. CONCLUSIONS: Pure-tone threshold testing incorporating high frequencies detects adolescent hearing loss more often than rapid hearing screens. Most state hearing screens omit high-frequency testing, potentially missing high-frequency losses, such as noise-induced hearing loss. Because noise-induced hearing loss in particular is preventable and hazardous noise exposures have increased, a reliable school hearing screen to detect high-frequency hearing loss in adolescents is warranted.