The role of cancer cell bioenergetics in dormancy and drug resistance.

While anti-cancer drug treatments are often effective for the clinical management of cancer, these treatments frequently leave behind drug-tolerant persister cancer cells that can ultimately give rise to recurrent disease. Such persistent cancer cells can lie dormant for extended periods of time, going undetected by conventional clinical means. Understanding the mechanisms that such dormant cancer cells use to survive, and the mechanisms that drive emergence from dormancy, is critical to the development of improved therapeutic strategies to prevent and manage disease recurrence. Cancer cells often exhibit metabolic alterations compared to their non-transformed counterparts. An emerging body of evidence supports the notion that dormant cancer cells also have unique metabolic adaptations that may offer therapeutically targetable vulnerabilities. Herein, we review mechanisms through which cancer cells metabolically adapt to persist during drug treatments and develop drug resistance. We also highlight emerging therapeutic strategies to target dormant cancer cells via their metabolic features.

Tumour, whole-blood, plasma and tissue concentrations of metformin in lung cancer patients.

AIM: Metformin is used for the management of type 2 diabetes mellitus (T2DM) and is being tested clinically as an anticancer agent. Metformin concentrations safely achievable in human solid tissues including tumours are unknown. This study was designed to determine metformin concentration in tissue compartments as a function of dose to inform rational dosing in preclinical models and interpretation of clinical results." METHODS: Subjects with solid tumours to be treated by resection and either (A) willingness to take metformin for 7-10 days before surgery or (B) taking metformin for T2DM were eligible. Whole blood, plasma, tumour, tumour-adjacent uninvolved tissue and subcutaneous adipose tissue were obtained for liquid chromatography with tandem mass spectrometry to measure metformin concentrations. RESULTS: All subjects had primary lung tumours. Metformin dose was significantly correlated with drug concentrations in all tissues analysed. Intersubject metformin concentrations varied by over two orders of magnitude. Metformin concentrations were significantly higher in tumour tissues and lower in adipose tissues compared to other tissues. Concentrations in blood and plasma were significantly correlated with concentrations in solid tissues. CONCLUSION: Metformin accumulates in cellular compartments. Concentrations observed in plasma, blood, lung and tumour tissues in subjects treated with US Food and Drug Administration-approved doses for T2DM are lower than those typically used in tissue culture studies. However, such tissue concentrations are in line with those found within cultured cells treated with supra-pharmacological doses of metformin. Given the large intersubject variability in metformin concentrations, it is imperative to determine whether there is an association between tissue metformin concentration and anticancer activity in humans.

The effect of botulinum toxin on ureteral inflammation.

PURPOSE: The impact of onabotulinum toxin type A (BoNT-A) on bladder afferent nerve pathways and chemosensory functions is an active area of investigation. There may be a role for BoNT-A in disorders of the ureter; however, no histologic studies have assessed the effects of BoNT-A on ureteral tissue. Our objective was to develop an animal model of ureteral inflammation and determine the impact of ureteral BoNT-A instillation on known mechanisms of inflammation. METHODS: The safety and feasibility of a novel animal model of ureteral inflammation was assessed. Through open cystotomy, the effect of ureteral BoNT-A instillation on inflammation was determined through H&E, masson's trichrome, Ki-67 stain, and prostaglandin E (PGE) synthase expression, a known marker of pain and inflammation in ureteral tissue. Urothelial microstructure was assessed using electron microscopy and standard histologic techniques. RESULTS: All experiments were carried to completion, and no systemic signs of botulinum toxicity were seen. BoNT-A exposure was associated with a decrease in PGE synthase expression in a dose-dependent fashion. BoNT-A exposure was not found to impact collagen deposition or cell proliferation. Disruption of tight junctions between urothelial cells was observed under conditions of inflammation. CONCLUSION: We describe the feasibility of a novel in vivo model of ureteral inflammation and report the first histologic study of the effects of BoNT-A on the ureter. Preliminary findings show that BoNT-A attenuates ureteral PGE synthase expression under conditions of inflammation. The application of BoNT-A may provide anti-inflammatory and analgesic effects in the context of ureteral disorders.

Immunogenetic effects of low dose (CEM43 30) magnetic nanoparticle hyperthermia and radiation in melanoma cells.

Objective: In this in vitro study we have used an RNA quantification technique, nanoString, and a conventional protein analysis technique (Western Blot) to assess the genetic and protein expression of B16 murine melanoma cells following a modest magnetic nanoparticle hyperthermia (mNPH) dose equivalent to 30 minutes @ 43°C (CEM43 30) and/or a clinically relevant 8 Gy radiation dose.Methods: Melanoma cells with mNPs(2.5 µg Fe/106 cells) were pelleted and exposed to an alternating magnetic field (AMF) to generate the targeted thermal dose. Thermal dose was accurately monitored by a fiber optic probe and automatically maintained at CEM43 30. All cells were harvested 24 hours after treatment.Results: The mNPH dose demonstrated notable elevations in the thermotolerance/immunogenic HSP70 gene and a number of chemoattractant and toll-like receptor gene pathways. The 8 Gy dose also upregulated a number of important immune and cytotoxic genetic and protein pathways. However, the mNPH/radiation combination was the most effective stimulator of a wide variety of immune and cytotoxic genes including HSP70, cancer regulating chemokines CXCL10, CXCL11, the T-cell trafficking chemokine CXCR3, innate immune activators TLR3, TLR4, the MDM2 and mTOR negative regulator of p53, the pro-apoptotic protein PUMA, and the cell death receptor Fas. Importantly a number of the genetic changes were accurately validated by protein expression changes, i.e., HSP70, p-mTOR, p-MDM2.Conclusion: These results not only show that low dose mNPH and radiation independently increase the expression of important immune and cytotoxic genes but that the effect is greatly enhanced when they are used in combination.

Alpelisib Efficacy without Cherry-PI3King Mutations.

The PI3K inhibitor alpelisib is clinically approved for the treatment of metastatic estrogen receptor-positive breast cancers harboring hotspot mutations in PIK3CA, which encodes a subunit of PI3K. Prospective clinical trial results demonstrated benefit from alpelisib for the treatment of advanced ER+ breast cancers harboring PIK3CA mutations in the hotspots of exons 7, 9, and 20. However, 20% of PIK3CA mutations occur in non-hotspot regions. A recent article demonstrated that patients with cancers bearing non-hotspot PIK3CA mutations also derived benefit from alpelisib, which will inform clinical decision-making moving forward. See related article by Rugo et al., p. 1056.

Estrogen Therapy Induces Receptor-Dependent DNA Damage Enhanced by PARP Inhibition in ER+ Breast Cancer.

PURPOSE: Clinical evidence indicates that treatment with estrogens elicits anticancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor α (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains underused. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. EXPERIMENTAL DESIGN: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDX), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. RESULTS: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacologic suppression of the DNA damage response via PARP inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2-mutant and BRCA1/2-wild-type cell line and PDX models. CONCLUSIONS: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer.

Purpose: Clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. Experimental Design: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. Results: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2 -mutant and BRCA1 /2-wild-type cell line and PDX models. Conclusions: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Electrostatically Driven Guanidinium Interaction Domains that Control Hydrogel-Mediated Protein Delivery In Vivo.

Protein biologics are an important class of drugs, but the necessity for frequent parenteral administration is a major limitation. Drug-delivery materials offer a potential solution, but protein-material adsorption can cause denaturation, which reduces their effectiveness. Here, we describe a new protein delivery platform that limits direct contact between globular protein domains and material matrix, yet from a single subcutaneous administration can be tuned for long-term drug release. The strategy utilizes complementary electrostatic interactions made between a suite of designed interaction domains (IDs), installed onto the terminus of a protein of interest, and a negatively charged self-assembled fibrillar hydrogel. These intermolecular interactions can be easily modulated by choice of ID to control material interaction and desorption energies, which allows regulation of protein release kinetics to fit desired release profiles. Molecular dynamics studies provided a molecular-level understanding of the mechanisms that govern release and identified optimal binding zones on the gel fibrils that facilitate strong ID-material interactions, which are crucial for sustained release of protein. This delivery platform can be easily loaded with cargo, is shear-thin syringe implantable, provides improved protein stability, is capable of a diverse range of in vitro release rates, and most importantly, can accomplish long-term control over in vivo protein delivery.