Oxygen tension-dependent variability in the cancer cell kinome impacts signaling pathways and response to targeted therapies.

Most cells in solid tumors are exposed to oxygen levels between 0.5% and 5%. We developed an approach that allows collection, processing, and evaluation of cancer and non-cancer cells under physioxia, while preventing exposure to ambient air. This aided comparison of baseline and drug-induced changes in signaling pathways under physioxia and ambient oxygen. Using tumor cells from transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen-dependent differences in cell preference for epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor beta (PDGFRß) signaling. Physioxia caused PDGFRß-mediated activation of AKT and extracellular regulated kinase (ERK) that reduced sensitivity to EGFR and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) inhibition and maintained PDGFRß+ epithelial-mesenchymal hybrid cells with potential cancer stem cell (CSC) properties. Cells in ambient air displayed differential EGFR activation and were more sensitive to targeted therapies. Our data emphasize the importance of oxygen considerations in preclinical cancer research to identify effective drug targets and develop combination therapy regimens.

Mutant RAS-driven Secretome Causes Skeletal Muscle Defects in Breast Cancer.

Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple-negative breast cancer (TNBC), estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in approximately 37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Because RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects. SIGNIFICANCE: Mutant RAS- and PIK3CA-driven breast cancers distinctly affect the function of skeletal muscle. Therefore, research and therapeutic targeting of cancer-induced systemic effects need to take aberrant cancer genome into consideration.

Modeling Preclinical Cancer Studies under Physioxia to Enhance Clinical Translation.

Oxygen (O2) plays a key role in cellular homeostasis. O2 levels are tightly regulated in vivo such that each tissue receives an optimal amount to maintain physiologic status. Physiologic O2 levels in various organs range between 2% and 9% in vivo, with the highest levels of 9% in the kidneys and the lowest of 0.5% in parts of the brain. This physiologic range of O2 tensions is disrupted in pathologic conditions such as cancer, where it can reach as low as 0.5%. Regardless of the state, O2 tension in vivo is maintained at significantly lower levels than ambient O2, which is approximately 21%. Yet, routine in vitro cellular manipulations are carried out in ambient air, regardless of whether or not they are eventually transferred to hypoxic conditions for subsequent studies. Even brief exposure of hematopoietic stem cells to ambient air can cause detrimental effects through a mechanism termed extraphysiologic oxygen shock/stress (EPHOSS), leading to reduced engraftment capabilities. Here, we provide an overview of the effects of ambient air exposure on stem and non-stem cell subtypes, with a focus on recent findings that reveal the impact of EPHOSS on cancer cells.

Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity.

Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O2. Here, we demonstrate that tumors collected, processed, and propagated at physiologic O2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Nexus between PI3K/AKT and Estrogen Receptor Signaling in Breast Cancer.

Signaling from estrogen receptor alpha (ERα) and its ligand estradiol (E2) is critical for growth of ≈70% of breast cancers. Therefore, several drugs that inhibit ERα functions have been in clinical use for decades and new classes of anti-estrogens are continuously being developed. Although a significant number of ERα+ breast cancers respond to anti-estrogen therapy, ≈30% of these breast cancers recur, sometimes even after 20 years of initial diagnosis. Mechanism of resistance to anti-estrogens is one of the intensely studied disciplines in breast cancer. Several mechanisms have been proposed including mutations in ESR1, crosstalk between growth factor and ERα signaling, and interplay between cell cycle machinery and ERα signaling. ESR1 mutations as well as crosstalk with other signaling networks lead to ligand independent activation of ERα thus rendering anti-estrogens ineffective, particularly when treatment involved anti-estrogens that do not degrade ERα. As a result of these studies, several therapies that combine anti-estrogens that degrade ERα with PI3K/AKT/mTOR inhibitors targeting growth factor signaling or CDK4/6 inhibitors targeting cell cycle machinery are used clinically to treat recurrent ERα+ breast cancers. In this review, we discuss the nexus between ERα-PI3K/AKT/mTOR pathways and how understanding of this nexus has helped to develop combination therapies.

Clofibrate, a PPAR-α agonist, abrogates sodium fluoride-induced neuroinflammation, oxidative stress, and motor incoordination via modulation of GFAP/Iba-1/anti-calbindin signaling pathways.

Fluoride is an environmental contaminant that is ubiquitously present in air, water, and soil. It is commonly added in minute quantity to drinking water, toothpaste, and mouth rinses to prevent tooth decay. Epidemiological findings have demonstrated that exposure to fluoride induced neurodevelopmental toxicity, developmental neurotoxicity, and motor disorders. The neuroprotective effect of clofibrate, a peroxisome proliferator-activated receptor alpha agonist, was investigated in the present study. Forty male Wistar rats were used for this study and randomly grouped into 10 rats per group as control, sodium fluoride (NaF) alone (300 ppm), NaF plus clofibrate (250 mg/kg), and NaF plus lisinopril (10 mg/kg), respectively, for 7 days. NaF was administered in drinking water while clofibrate and lisinopril were administered by oral gavage. Markers of neuronal inflammation and oxidative stress, acetylcholinesterase activity, and neurobehavioral (hanging wire and open field) tests were performed. Immunohistochemistry was performed on brain tissues, and they were probed with glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, and cerebellar Ca2+ -binding protein calbindin-D28k. The results showed that NaF significantly increased of oxidative stress and neuroinflammation and inhibited AChE activity. Immunostaining showed reactive astrocytes, microgliosis, loss of dendritic spines, and arborization in Purkinje cells in rats administered only NaF. Neurobehavioral results showed that cotreatment of NaF with clofibrate improved muscular strength and locomotion, reduced anxiety, and significantly reduced astrocytic count. Overall, cotreatment of NaF with either clofibrate or lisinopril showed neuroprotective effects by mitigating neuronal inflammation and oxidative and motor incoordination. Hence, clofibrate could be seen as a novel drug candidate against neurodegeneration and motor disorders.

Characterisation of heart failure with normal ejection fraction in a tertiary hospital in Nigeria.

BACKGROUND: The study aimed to determine the frequency and characteristics of heart failure with normal EF in a native African population with heart failure. METHODS: It was a hospital cohort study. Subjects were 177 consecutive individuals with heart failure and ninety apparently normal control subjects. All the subjects underwent transthoracic echocardiography. The group with heart failure was further subdivided into heart failure with normal EF (EF > or = 50) (HFNEF) and heart failure with low EF(EF <50)(HFLEF). RESULTS: The subjects with heart failure have a mean age of 52.3 +/- 16.64 years vs 52.1 +/- 11.84 years in the control subjects; p = 0.914. Other baseline characteristics except blood pressure parameters and height were comparable between the group with heart failure and the control subjects. The frequency of HFNEF was 39.5%. Compared with the HFLEF group, the HFNEF group have a smaller left ventricular diameter (in diastole and systole): (5.2 +/- 1.22 cm vs 6.2 +/- 1.39 cm; p < 0.0001 and 3.6 +/- 1.24 cm vs 5.4 +/- 1.35 cm;p < 0.0001) respectively, a higher relative wall thickness and deceleration time of the early mitral inflow velocity: (0.4 +/- 0.12 vs 0.3 +/- 0.14 p < 0.0001 and 149.6 +/- 72.35 vs 110.9 +/- 63.40 p = 0.001) respectively. The two groups with heart failure differed significantly from the control subjects in virtually all echocardiographic measurements except aortic root diameter, LV posterior wall thickness(HFLEF), and late mitral inflow velocity(HFNEF). HFNEF accounted for 70(39.5%) of cases of heart failure in this study. Hypertension is the underlying cardiovascular disease in 134(75.7%) of the combined heart failure population, 58 (82.9%) of the subjects with HFNEF group and 76(71%) of the HFLEF group. Females accounted for 44 (62.9%) of the subjects with HFNEF against 42(39.3%) in the HFLEF group (p = 0.002). CONCLUSION: The frequency of heart failure with normal EF in this native African cohort with heart failure is comparable with the frequency in other populations. These groups of patients are more likely female, hypertensive with concentric pattern of left ventricular hypertrophy.

Blood pressure control and left ventricular hypertrophy in hypertensive Nigerians.

BACKGROUND: Hypertension is a disease characterized by end-organ complications, leading to high morbidity and mortality in many cases. People with untreated or uncontrolled hypertension often run the risk of developing complications directly associated with the disease. Left ventricular hypertrophy (LVH) has been shown to be a significant risk factor for adverse outcomes both in patients with hypertension and in the general population. We investigated the prevalence and pattern of LVH in a treated hypertensive population at the University College Hospital, Ibadan, Nigeria, using non-hypertensive subjects as control. Design and Setting : A prospective observational study performed at the University College Hospital, Ibadan, Nigeria. METHODS: Patients had 6 visits, when at least one blood pressure measurement was recorded for each hypertensive subject and average calculated for systolic blood pressure (SBP) and diastolic blood pressure (DBP) separately. The values obtained were used for stratification of the subjects into controlled and uncontrolled hypertension. Subjects also had echocardiograms to determine their left ventricular mass. RESULTS: LVH was found in 14 (18.2%) of the normotensive group, 40 (20.8%) of the uncontrolled hypertensive group and 14 (24.1%) of the controlled hypertensive group when left ventricular mass (LVM) was indexed to body surface area (BSA). When LVM was indexed to height, left ventricular hypertrophy was found in none of the subjects of the normotensive group, while it was found present in 43 (22.4%) and 14 (24.1%) subjects of the uncontrolled and controlled hypertensive groups, respectively. Significant difference in the prevalence of LVH was detected only when LVM was indexed to height alone. CONCLUSION: Clinic blood pressure is an ineffective way of assessing BP control. Thus in apparently controlled hypertensive subjects, based on office blood pressure, cardiac structural changes do remain despite antihypertensive therapy. This population is still at risk of cardiovascular events.

Left ventricular geometric patterns in normotensive type 2 diabetic patients in Nigeria: an echocardiographic study.

Left ventricular hypertrophy has been linked with diabetes mellitus and abnormal glucose tolerance in several studies. Most previous studies have been carried out in the western world with dearth of data in native Africans. A total of 122 type 2 diabetic patients with a mean age of 55.0+/-8.5 years and another 90 normal patients with a mean age of 55.4+/-8.7 years were recruited for the study. Two-dimensional guided M-mode echocardiography was performed on each patient. In the diabetic patients, 49.2% had normal geometry, 23.0% had concentric hypertrophy, 13.0% had concentric remodeling, and 14.8% had eccentric hypertrophy. In the control group, 72.2% had normal geometry, 4.4% had concentric hypertrophy, 11.2% had concentric remodeling, and 12.2% had eccentric hypertrophy. In a multiple regression analysis, there was significant difference in the geometric pattern of the diabetics and controls (chi(2)=11.09, P<.001). Diabetes mellitus is independently associated with left ventricular structural changes in Nigerian diabetics.

Characterisation of left ventricular function by tissue Doppler imaging technique in newly diagnosed, untreated hypertensive subjects.

BACKGROUND: Hypertension results in structural and functional changes in the heart. Early detection of abnormalities of cardiac structure and function is important in the assessment and treatment of hypertensive subjects. The aim of this study was to evaluate the utility of the tissue Doppler echocardiographic technique in characterising diastolic and systolic functions in untreated native black African hypertensive subjects. MATERIALS AND METHODS: Forty consecutive, newly diagnosed, untreated hypertensives with adequate conventional echocardiographic (2-D, M-mode, transmitral and pulmonary Doppler flow velocities) and tissue Doppler echocardiographic images were recruited into the study. The control subjects were apparently normal individuals. Each arm of the study consisted of 21 male and 19 female subjects. RESULTS: The two groups were comparable by age (48.6 +/- 11.35 years in the hypertensives vs 48.1 +/- 11.33 years in the controls; p = 0.844) and gender distribution (M/F: 21/19 in both groups). Other baseline characteristics, except for blood pressure parameters, which were predictably higher in the hypertensive subjects, were comparable between the two groups. The hypertensive subjects had a lower systolic myocardial velocity (Sm) and early diastolic myocardial velocity (Em) in comparison with the controls (p = 0.033 and p = 0.018, respectively). The late diastolic myocardial velocity (Am) was comparable in the two groups (p = 0.430). CONCLUSIONS: Tissue Doppler echocardiography demonstrates diastolic dysfunction relatively early in native African hypertensives and may be useful for detecting subtle deterioration in systolic function.

Changes in left atrial dimension and function and left ventricular geometry in newly diagnosed untreated hypertensive subjects.

BACKGROUND: It is known that a spectrum of changes in structure, size and function of the different chambers of the heart occur in individuals with hypertension. The earliest changes and the sequence of these changes are still being studied. AIMS: The present study aimed to assess early changes in the left atrial size and function in hypertension, and its relationship with left ventricular geometry and other factors that may influence left atrial size. METHODS: One hundred consecutive subjects who were newly diagnosed with hypertension and 50 apparently normal individuals were recruited into the study. Standard M-mode, two-dimensional and Doppler echocardiography were performed. The endocardial border of the left atrium was traced to obtain the atrial area and left atrial volumes and emptying fractions were derived from measured areas. RESULTS: The hypertensive patients and the controls were comparable by age, sex and body mass index. Thirty-seven (37%) of the hypertensive subject had increased left ventricular mass versus eight subjects (16%) in the normal controls. The patients with hypertension had a higher linear left atrial dimension (3.5 +/- 0.48 cm versus 3.1 +/- 0.47 cm, P < 0.0001), longer pre-atrial contraction length (3.8 +/- 0.56 cm versus 3.6 +/- 0.45 cm; P = 0.02) and higher peak late mitral inflow velocity (0.64 +/- 0.19 m/s versus 0.56 +/- 0.15 m/s; P = 0.010). CONCLUSIONS: Changes in the geometry of the left ventricle occur early in hypertension and precede deterioration in left ventricular systolic function. The corresponding left atrial changes are marginal and are indicative of increased left atrial length and accentuated atrial systolic function.

Characterisation of left ventricular function by tissue Doppler imaging technique in newly diagnosed, untreated hypertensive subjects

BACKGROUND:Hypertension results in structural and functional changes in the heart. Early detection of abnormalities of cardiac structure and function is important in the assessment and treatment of hypertensive subjects. The aim of this study was to evaluate the utility of the tissue Doppler echocardiographic technique in characterising diastolic and systolic functions in untreated native black African hypertensive subjects.MATERIALS AND METHODS:Forty consecutive, newly diagnosed, untreated hypertensives with adequate conventional echocardiographic (2-D, M-mode, transmitral and pulmonary Doppler flow velocities) and tissue Doppler echocardiographic images were recruited into the study. The control subjects were apparently normal individuals. Each arm of the study consisted of 21 male and 19 female subjects.RESULTS:The two groups were comparable by age (48.6 +/- 11.35 years in the hypertensives vs 48.1 +/- 11.33 years in the controls; p = 0.844) and gender distribution (M/F: 21/19 in both groups). Other baseline characteristics, except for blood pressure parameters, which were predictably higher in the hypertensive subjects, were comparable between the two groups. The hypertensive subjects had a lower systolic myocardial velocity (Sm) and early diastolic myocardial velocity (Em) in comparison with the controls (p = 0.033 and p = 0.018, respectively). The late diastolic myocardial velocity (Am) was comparable in the two groups (p = 0.430). CONCLUSIONS:Tissue Doppler echocardiography demonstrates diastolic dysfunction relatively early in native African hypertensives and may be useful for detecting subtle deterioration in systolic function

Echocardiographic partition values and prevalence of left ventricular hypertrophy in hypertensive Nigerians.

BACKGROUND: Left ventricular hypertrophy (LVH) is a well known independent risk factor for cardiovascular events. It has been shown that combination of left ventricular mass (LVM) and relative wall thickness (RWT) can be used to identify different forms of left ventricular (LV) geometry. Prospective studies have shown that LV geometric patterns have prognostic implications, with the worst prognosis associated with concentric hypertrophy. The methods for the normalization or indexation of LVM have also recently been shown to confer some prognostic value especially in obese population. We sought to determine the prevalence of echocardiographic lLVH using eight different and published cut-off or threshold values in hypertensive subjects seen in a developing country's tertiary centre. METHODS: Echocardiography was performed in four hundred and eighty consecutive hypertensive subjects attending the cardiology clinic of the University college Hospital Ibadan, Nigeria over a two-year period. RESULTS: Complete data was obtained in 457 (95.2%) of the 480 subjects (48.6% women). The prevalence of LVH ranged between 30.9-56.0%. The highest prevalence was when LVM was indexed to the power of 2.7 with a partition value of 49.2 g/ht2.7 in men and 46.7 g/ht2.7 in women. The lowest prevalence was observed when LVM was indexed to body surface area (BSA) and a partition value of 125 g/m2 was used for both sexes. Abnormal LV geometry was present in 61.1%-74.0% of our subjects and commoner in women. CONCLUSION: The prevalence of LVH hypertensive patients is strongly dependent on the cut-off value used to define it. Large-scale prospective study will be needed to determine the prognostic implications of the different LV geometry in native Africans.