9S1R nullomer peptide induces mitochondrial pathology, metabolic suppression, and enhanced immune cell infiltration, in triple-negative breast cancer mouse model.

Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide prime sequence attached to 5-arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo mouse model. This unique peptide, administered with a trehalose carrier (9S1R-NulloPT), offers enhanced solubility and exhibits distinct anti-cancer effects against TNBC. In our study, we investigated the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, tumor immune-microenvironment (TME), and transcriptome of aggressive mouse TNBC tumors. Notably, treated mice had smaller tumors in the initial phase of the treatment, as compared to untreated control, and diminished in vivo and ex vivo bioluminescence at later-stages - indicative of metabolically quiescent, dying tumors. The treatment also caused changes in TME with increased infiltration of immune cells and altered tumor transcriptome, with 365 upregulated genes and 710 downregulated genes. Consistent with in vitro data, downregulated genes were enriched in cellular metabolic processes (179), specifically mitochondrial TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis (45). The upregulated genes were associated with the developmental (13), ECM organization (12) and focal adhesion pathways (7). In conclusion, our study demonstrates that 9S1R-NulloPT effectively reduced tumor growth during its initial phase, altering the TME and tumor transcriptome. The treatment induced mitochondrial pathology which led to a metabolic deceleration in tumors, aligning with in vitro observations.

Cardiopulmonary Exercise Testing in a Prospective Multicenter Cohort of Older Adults.

PURPOSE: Cardiorespiratory fitness (CRF) measured by peak oxygen consumption (V̇O 2peak ) declines with aging and correlates with mortality and morbidity. Cardiopulmonary exercise testing (CPET) is the criterion method to assess CRF, but its feasibility, validity, and reliability in older adults are unclear. Our objective was to design and implement a dependable, safe, and reliable CPET protocol in older adults. METHODS: V̇O 2peak was measured by CPET, performed using treadmill exercise in 875 adults ≥70 yr in the Study of Muscle, Mobility and Aging (SOMMA). The protocol included a symptom-limited peak (maximal) exercise and two submaximal walking speeds. An adjudication process was in place to review tests for validity if they met any prespecified criteria (V̇O 2peak <12.0 mL·kg -1 ·min -1 ; maximum heart rate <100 bpm; respiratory exchange ratio <1.05 and a rating of perceived exertion <15). A subset ( N = 30) performed a repeat test to assess reproducibility. RESULTS: CPET was safe and well tolerated, with 95.8% of participants able to complete the V̇O 2peak phase of the protocol. Only 56 (6.4%) participants had a risk alert and only two adverse events occurred: a fall and atrial fibrillation. Mean ± SD V̇O 2peak was 20.2 ± 4.8 mL·kg -1 ·min -1 , peak heart rate 142 ± 18 bpm, and peak respiratory exchange ratio 1.14 ± 0.09. Adjudication was indicated in 47 tests; 20 were evaluated as valid and 27 as invalid (18 data collection errors, 9 did not reach V̇O 2peak ). Reproducibility of V̇O 2peak was high (intraclass correlation coefficient = 0.97). CONCLUSIONS: CPET was feasible, effective, and safe for older adults, including many with multimorbidity or frailty. These data support a broader implementation of CPET to provide insight into the role of CRF and its underlying determinants of aging and age-related conditions.

The role of maximal inspiratory pressure on functional performance in adults with heart failure.

BACKGROUND: Exercise intolerance is common among adults with heart failure (HF) and is a strong prognostic indicator. We examined maximal inspiratory pressure (MIP) as an indicator of maximal and submaximal exercise capacity in older HF patients. METHODS: Fifty-one patients age ≥ 50 years with HF underwent MIP testing via the PrO2 device. Peak oxygen uptake (VO2), 6 min walk distance (6MWD), 30 s sit-to-stand test (STS), gait speed (GS), grip strength and lower extremity muscle strength [one-repetition maximum (1RM)] were measured. Correlation and exploratory multiple regression analyses investigated relationships between MIP, left ventricular ejection fraction (LVEF), age, body mass index (BMI) and physical function. MIP was then stratified by median (64 cm H2O), and endpoints were compared between median groups. RESULTS: The median age was 69 years [interquartile range (IQR): 66-73], and the median LVEF was 36.5% (IQR: 30%-45%). Regression identified MIP as an independent predictor for grip strength, 6MWD, 1RM weight and 30 s STS after adjustment for age, BMI and LVEF. MIP greater than the median (n = 25) independently predicted and reflected greater peak VO2 [14.2 (12.8-18.1) vs. 11.5 (9.7-13.0) mL/kg/min; P = 0.0007] as well as 6MWD, 1RM, 30 s STS and GS (all P < 0.05). CONCLUSION: The analysis demonstrates that MIP is a novel biometric for exercise tolerance in adults with HF. Assessments of MIP are safe and convenient, with the potential to enhance routine HF surveillance and provide novel biometrics to guide HF therapeutics.

The clinical relevance of OSM in inflammatory diseases: a comprehensive review.

Oncostatin M (OSM) is a pleiotropic cytokine involved in a variety of inflammatory responses such as wound healing, liver regeneration, and bone remodeling. As a member of the interleukin-6 (IL-6) family of cytokines, OSM binds the shared receptor gp130, recruits either OSMRß or LIFRß, and activates a variety of signaling pathways including the JAK/STAT, MAPK, JNK, and PI3K/AKT pathways. Since its discovery in 1986, OSM has been identified as a significant contributor to a multitude of inflammatory diseases, including arthritis, inflammatory bowel disease, lung and skin disease, cardiovascular disease, and most recently, COVID-19. Additionally, OSM has also been extensively studied in the context of several cancer types including breast, cervical, ovarian, testicular, colon and gastrointestinal, brain,lung, skin, as well as other cancers. While OSM has been recognized as a significant contributor for each of these diseases, and studies have shown OSM inhibition is effective at treating or reducing symptoms, very few therapeutics have succeeded into clinical trials, and none have yet been approved by the FDA for treatment. In this review, we outline the role OSM plays in a variety of inflammatory diseases, including cancer, and outline the previous and current strategies for developing an inhibitor for OSM signaling.

Nullomer peptide increases immune cell infiltration and reduces tumor metabolism in triple negative breast cancer mouse model.

Background: Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide derived from a prime sequence that is tagged with 5 arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo TNBC mouse model. 9S1R is administered in trehalose (9S1R-NulloPT), which enhances solubility and exhibits some independent effects against tumor growth and is thus an important component in the drug preparation. Method: We examined the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, necrosis, tumor immune microenvironment, and the transcriptome of aggressive mouse TNBC tumors. Results: The peptide-treated mice had smaller tumors in the initial phase of the treatment, as compared to the untreated control, and reduced in vivo bioluminescence at later stages, which is indicative of metabolically inactive tumors. A decrease in ex vivo bioluminescence was also observed in the excised tumors of treated mice, but not in the secondary metastasis in the lungs. The treatment also caused changes in tumor immune microenvironment with increased infiltration of immune cells and margin inflammation. The treatment upregulated 365 genes and downregulated 710 genes in tumors compared to the untreated group. Consistent with in vitro findings in breast cancer cell lines, downregulated genes in the treated TNBC tumors include Cellular Metabolic Process Related genes (179), specifically mitochondrial genes associated with TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis genes (45). Among upregulated genes, the Developmental Pathway (13), ECM Organization (12) and Focal Adhesion Related Pathways (7) were noteworthy. We also present data from a pilot study using a bilateral BC mouse model, which supports our findings. Conclusion: In conclusion, although 9S1R-NulloPT was moderate at reducing the tumor volume, it altered the tumor immune microenvironment as well as the tumor transcriptome, rendering tumors metabolically less active by downregulating the mitochondrial function and ribosome biogenesis. This corroborates previously published in vitro findings.

Cardiopulmonary Exercise Testing in a Prospective Multicenter Cohort of Older Adults: The Study of Muscle, Mobility and Aging (SOMMA).

BACKGROUND: Cardiorespiratory fitness (CRF) measured by peak oxygen consumption (VO2peak) declines with aging and correlates with mortality and morbidity. Cardiopulmonary Exercise Testing (CPET) has long been the criterion method to assess CRF, but its feasibility, efficacy and reliability in older adults is unclear. The large, multicenter Study of Muscle, Mobility and Aging (SOMMA) employed CPET to evaluate the mechanisms underlying declines in mobility with aging among community-dwelling older adults. Our primary objective was to design and implement a CPET protocol in older adults that was dependable, safe, scientifically valuable, and methodologically reliable. METHODS: CPET was performed using treadmill exercise in 875 adults ≥70 years. A composite protocol included a symptom-limited peak exercise phase and two submaximal phases to assess cardiopulmonary ventilatory indices during 1) participants' preferred walking speed and 2) at slow walking speed of 1.5 mph (0.67 m/s). An adjudication process was in place to review tests for validity if they met any prespecified criteria (VO2peak <12.0 ml/kg/min; maximum heart rate (HR) <100 bpm; respiratory exchange ratio (RER) <1.05 and a rating of perceived exertion <15). A repeat test was performed in a subset (N=30) to assess reproducibility. RESULTS: CPET was safe and well tolerated, with 95.8% of participants able to complete the VO2peak phase of the protocol. Only 56 (6.4%) participants had a risk alert during any phase of testing and only two adverse events occurred during the peak phase: a fall and atrial fibrillation. The average ± standard deviation for VO2peak was 20.2 ± 4.8 mL/kg/min, peak HR 142 ± 18 bpm, and peak RER 1.14 ± 0.09. VO2peak and RER were slightly higher in men than women. Adjudication was indicated in 47 participants; 20 were evaluated as valid, 27 as invalid (18 had a data collection error, 9 did not reach VO2peak). Reproducibility of VO2peak was high (intraclass correlation coefficient=0.97). CONCLUSIONS: CPET was feasible, effective and safe for community-dwelling older adults, many of whom had multimorbidity and frailty. These data support a broader implementation of CPET to provide important insight into the role of CRF and its underlying determinants in aging and age-related conditions and diseases. Clinical Perspective: What Is New?: Performing cardiopulmonary exercise testing in a community dwelling older adult with multimorbidities or frailty is feasible and exceptionally safe under highly trained exercise physiologists and physician supervision.Reproducibility of VO2peak among community-dwelling older adults with significant clinical complexity was high (intraclass correlation coefficient=0.97).The VO2peak observed was comparable to established normative data for older adults, and adds merit to the limited data collected on VO2peak norms in older adults.What Are the Clinical Implications?: Ventilatory gas collection during clinical cardiac stress testing may be valuable to plan of care in routine management of older adults due to the important role of aerobic fitness on morbidity and mortality.Cardiopulmonary exercise testing can provide insight into the role of cardiorespiratory fitness and its underlying determinants in aging and age-related conditions and diseases.

Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery.

Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 µM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening.

HIGH expression of OSM and IL-6 are associated with decreased breast cancer survival: synergistic induction of IL-6 secretion by OSM and IL-1ß.

Chronic inflammation has been recognized as a risk factor for the development and maintenance of malignant disease. Cytokines such as interleukin-6 (IL-6), oncostatin M (OSM), and interleukin-1 beta (IL-1ß) promote the development of both acute and chronic inflammation while promoting in vitro metrics of breast cancer metastasis. However, anti-IL-6 and anti-IL-1ß therapeutics have not yielded significant results against solid tumors in clinical trials. Here we show that these three cytokines are interrelated in expression. Using the Curtis TCGA™ dataset, we have determined that there is a correlation between expression levels of OSM, IL-6, and IL-1ß and reduced breast cancer patient survival (r = 0.6, p = 2.2 x 10-23). Importantly, we confirm that OSM induces at least a 4-fold increase in IL-6 production from estrogen receptor-negative (ER-) breast cancer cells in a manner that is dependent on STAT3 signaling. Furthermore, OSM induces STAT3 phosphorylation and IL-1ß promotes p65 phosphorylation to synergistically induce IL-6 secretion in ER- MDA-MB-231 and to a lesser extent in ER+ MCF7 human breast cancer cells. Induction may be reduced in the ER+ MCF7 cells due to a previously known suppressive interaction between ER and STAT3. Interestingly, we show in MCF7 cells that ER's interaction with STAT3 is reduced by 50% through both OSM and IL-1ß treatment, suggesting a role for ER in mitigating STAT3-mediated inflammatory cascades. Here, we provide a rationale for a breast cancer treatment regime that simultaneously suppresses multiple targets, as these cytokines possess many overlapping functions that increase metastasis and worsen patient survival.

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition.

BACKGROUND: Hormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT). METHODS: The relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells. RESULTS: ER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don't detach at significant rates. CONCLUSION: OSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.