Small-molecule modulators of mitochondrial channels as chemotherapeutic agents

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met

Abstract Introduction Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC). Methods The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student\u27s t test was used for all statistical analysis. Results Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI. Conclusions Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions

Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling.

Matriptase is an epithelia-specific membrane-anchored serine protease that has received considerable attention in recent years because of its consistent dysregulation in human epithelial tumours, including breast cancer. Mice with reduced levels of matriptase display a significant delay in oncogene-induced mammary tumour formation and blunted tumour growth. The abated tumour growth is associated with a decrease in cancer cell proliferation. Here we demonstrate by genetic deletion and silencing that the proliferation impairment in matriptase-deficient breast cancer cells is caused by their inability to initiate activation of the c-Met signalling pathway in response to fibroblast-secreted pro-HGF. Similarly, inhibition of matriptase catalytic activity using a selective small-molecule inhibitor abrogates the activation of c-Met, Gab1 and AKT, in response to pro-HGF, which functionally leads to attenuated proliferation in breast carcinoma cells. We conclude that matriptase is critically involved in breast cancer progression and represents a potential therapeutic target in breast cancer

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs

Increased risk of asthma at age 10 years for children sensitized to multiple allergens

BACKGROUND: Childhood sensitization patterns have been previously found to be related to variable risk of early life allergic disease in several birth cohorts. OBJECTIVE: To determine whether these risks persist into later childhood. METHODS: In the birth cohort of the Wayne County Health, Environment, Allergy and Asthma Longitudinal Study, previous latent class analysis based on sensitization to 10 allergens found the following 4 early life sensitization patterns at age 2 years: highly sensitized, milk/egg dominated, peanut and inhalant(s), and low to no sensitization. At an age 10 study-specific visit, children were evaluated by an allergist for current asthma and atopic dermatitis through a physical examination and interviews with the child and parent or guardian. Total and specific immunoglobulin E (IgE), spirometry, and methacholine challenge were also completed. RESULTS: Compared with children sensitized to none or 1 allergen, children sensitized to 4 or more food and inhalant allergens at age 2 had the highest risk of current asthma (relative risk [RR], 4.42; 95% confidence interval [CI], 2.58-7.59; P \u3c .001) and bronchial hyperresponsiveness (RR, 1.77; 95% CI, 1.29-2.42; P \u3c .001). In addition, they had the highest levels of total IgE (geometric mean, 800 IU/mL; 95% CI, 416-1536) among the 4 groups. Risk of current atopic dermatitis did not depend on pattern of sensitization but remained increased for children with any sensitization (RR, 2.23; 95% CI, 1.40-3.55; P \u3c .001). No differences in spirometry (forced expiratory volume in 1 second, forced expiratory flow between 25% and 75%, and forced expiratory volume in 1 second/forced vital capacity) were identified. CONCLUSION: The previously reported importance of a specific pattern of sensitization in early life (sensitization to ≥4 inhalant and food allergens) continues to be associated with an increased risk of asthma, bronchial hyperresponsiveness, and high total IgE at age 10 years

Infant Feeding Practices and Subsequent Dietary Patterns of School-Aged Children in a US Birth Cohort

BACKGROUND: Infant feeding practices are thought to shape food acceptance and preferences. However, few studies have evaluated whether these affect child diet later in life. OBJECTIVE: The study objective was to examine the association between infant feeding practices and dietary patterns (DPs) in school-aged children. DESIGN: A secondary analysis of data from a diverse prospective birth cohort with 10 years of follow-up (WHEALS [Wayne County Health Environment Allergy and Asthma Longitudinal Study]) was conducted. PARTICIPANTS/SETTING: Children from the WHEALS (Detroit, MI, born 2003 through 2007) who completed a food screener at age 10 years were included (471 of 1,258 original participants). MAIN OUTCOME MEASURES: The main outcome was DPs at age 10 years, identified using the Block Kids Food Screener. STATISTICAL ANALYSIS PERFORMED: Latent class analysis was applied for DP identification. Breastfeeding and age at solid food introduction were associated with DPs using a 3-step approach for latent class modeling based on multinomial logistic regression models. RESULTS: The following childhood DPs were identified: processed/energy-dense food (35%), variety plus high intake (41%), and healthy (24%). After weighting for loss to follow-up and covariate adjustment, compared with formula-fed children at 1 month, breastfed children had 0.41 times lower odds of the processed/energy-dense food DP vs the healthy DP (95% CI 0.14 to 1.25) and 0.53 times lower odds of the variety plus high intake DP (95% CI 0.17 to 1.61), neither of which were statistically significant. Results were similar, but more imprecise, for breastfeeding at 6 months. In addition, the association between age at solid food introduction and DP was nonsignificant, with each 1-month increase in age at solid food introduction associated with 0.81 times lower odds of the processed/energy-dense food DP relative to the healthy DP (95% CI 0.64 to 1.02). CONCLUSIONS: A significant association between early life feeding practices and dietary patterns at school age was not detected. Large studies with follow-up beyond early childhood that can also adjust for the multitude of potential confounders associated with breastfeeding are needed

Childhood Allergy and the NeOnatal Environment (CANOE) Research Protocol and Recruitment Redesign during the COVID-19 Pandemic

Rationale: Recruitment for research studies is a challenging endeavor that has been further complicated by the COVID-19 pandemic. While clinical research was temporarily halted due to the pandemic, it was hypothesized that study and recruitment restructuring would enable brisk enrollment when research resumed. Methods: A new NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) was launched in January 2019 across four sites to determine how pre-, peri-, and post-natal factors influence development of recurrent wheezing and atopic dermatitis. Study recruitment was halted for nine months due to the COVID-19 pandemic, during which recruitment and study procedures were redesigned. Results: Recruitment strategies were modified to limit in-person contact, shifting toward alternative HIPAA-compliant methods like clinician referrals, institutional social media, and telemedicine consenting. Protocol changes included reducing frequency of in-person visits, leveraging clinical care visits to collect bio-samples, expanded self-collection of samples at home, and posting study materials online. Recruitment rates range from 3-12 families per month per site. In-clinic recruitment with modifications for social distancing has been successful across all sites. Other successful strategies have included targeted social media posts, mailed letters, and email. Rates of consent have been similar across recruitment strategies and the implementation of multiple recruitment strategies has yielded the highest rates of ongoing consent and enrollment of mother-infant dyads. Conclusions: Study procedures that prioritize health and safety measures such as social distancing, study participant convenience, and diversification of recruitment strategies enable continued birth cohort recruitment and data collection while adhering to public health restrictions during the pandemic

Examining virtual research recruitment and participant diversity in a multi-center birth cohort, Childhood Allergy and the NeOnatal Environment (CANOE)

Rationale: Recruitment for a NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) stopped due to the COVID-19 pandemic. Redesign of study procedures emphasized virtual and socially distanced activities. We hypothesized that “virtual” recruitment methods (social media, websites, email) would surpass “traditional” methods (in-clinic, telephone, flyers/print materials) and increase enrollment of families from diverse backgrounds and communities. Methods: Pregnant women (n=439, target 500) were recruited from four academic medical centers in Detroit MI, Madison WI, Nashville TN, and St. Louis MO. We collected demographic and social information by questionnaires and examined race, ethnicity, age, parity, and employment status in relation to recruitment method using chi-square tests. Results: In-clinic and telephone recruitment comprised 55% of enrollment, followed by print materials (17%), and social media and email (15%). The cohort includes families self-identifying as Caucasian/White (63%), African American/Black (27%), Hispanic/Latino (3.3%), Asian (3.5%), and mixed races (1.2%). This reflects site demographics for White and Black patients, while other populations are not as well recruited into this cohort. Recruitment method success did not vary by race, ethnicity, maternal age, or employment status (p=ns for each comparison). Most (63%) multigravida mothers (9.1% of participants) were recruited in clinic, while primigravida participants were recruited more evenly via all methods. Conclusions: “Virtual” recruitment methods comprised a smaller proportion of cohort enrollment than hypothesized and study recruitment method did not vary by race/ethnicity; however, consideration of combined, varied, and novel recruitment methods may add to the development of best practices for more representative research study recruitment

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondria! energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/beta-catenin signaling in vitro in colon cancer cells and in vivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondria! ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondria! energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologie