Therapy-induced senescence in breast cancer: an overview.

Outcomes for women with breast cancer have improved dramatically in recent decades. However, many patients present with intrinsic drug resistance and others are initially sensitive to anti-cancer drugs but acquire resistance during the course of their treatment, leading to recurrence and/or metastasis. Drug therapy-induced senescence (TIS) is a form of drug resistance characterised by the induction of cell cycle arrest and the emergence of a senescence-associated secretory phenotype (SASP) that can develop in response to chemo- and targeted- therapies. A wide range of anticancer interventions can lead to cell cycle arrest and SASP induction, by inducing genotoxic stress, hyperactivation of signalling pathways or oxidative stress. TIS can be anti-tumorigenic in the short-term, but pro-tumorigenic in the long-term by creating a pro-inflammatory and immunosuppressive microenvironment. Moreover, the SASP can promote angiogenesis and epithelial-mesenchymal transition in neighbouring cells. In this review, we will describe the characteristics of TIS in breast cancer and detail the changes in phenotype that accompany its induction. We also discuss strategies for targeting senescent cancer cells in order to prevent or delay tumour recurrence.

JRK binds satellite III DNA and is necessary for the heat shock response.

JRK is a DNA-binding protein of the pogo superfamily of transposons, which includes the well-known centromere binding protein B (CENP-B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates ß-catenin-TCF activity but little is known of its cellular functions. Based on its homology to CENP-B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK-GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti-JRK antiserum we show that endogenous JRK co-localises with a subset of centromeres in non-stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat-shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

A systematic computational analysis of the endosomal recycling pathway in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive brain cancer in adults. The standard treatment is brutal and has changed little in 20 years, and more than 85% of patients will die within two years of their diagnosis. There is thus an urgent need to identify new drug targets and develop novel therapeutic strategies to increase survival and improve quality of life. Using publicly available genomics, transcriptomics and proteomics datasets, we compared the expression of endosomal recycling pathway regulators in non-tumour brain tissue with their expression in GBM. We found that key regulators of this pathway are dysregulated in GBM and their expression levels can be linked to survival outcomes. Further analysis of the differentially expressed endosomal recycling regulators allowed us to generate an 8-gene prognostic signature that can distinguish low-risk from high-risk GBM and potentially identify tumours that may benefit from treatment with endosomal recycling inhibitors. This study presents the first systematic analysis of the endosomal recycling pathway in glioblastoma and suggests it could be a promising target for the development of novel therapies and therapeutic strategies to improve outcomes for patients.

Endosomal recycling inhibitors downregulate estrogen receptor-alpha and synergise with endocrine therapies.

PURPOSE: Breast cancer (BC) accounts for roughly 30% of new cancers diagnosed in women each year; thus, this cancer type represents a substantial burden for people and health care systems. Despite the existence of effective therapies to treat BC, drug resistance remains a problem and is a major cause of treatment failure. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. Recent research indicates that inhibition of the endosomal recycling pathway, an intracellular membrane trafficking pathway that returns endocytosed proteins back to the plasma membrane, may be a promising strategy to downregulate clinically relevant cell surface proteins such as HER2 and HER3, and to overcome drug resistance. METHODS: To investigate the molecular mechanism of action of an endosomal recycling inhibitor (ERI) called primaquine, we performed a reverse-phase protein array (RPPA) assay using a HER2-positive breast cancer cell line. The RPPA findings were confirmed by Western blot and RT-qPCR in several BC cell lines. Novel drug combinations were tested by MTT cell viability and clonogenic assays. RESULTS: Among the signalling molecules downregulated by ERIs were estrogen receptor-alpha (ER-α) and androgen receptor. We confirmed this finding in other breast cancer cell lines and show that downregulation occurs at the transcriptional level. We also found that ERIs synergise with tamoxifen, a standard-of-care therapy for breast cancer. DISCUSSION: Our data suggest that combining ERIs with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Endosomal recycling inhibitors downregulate the androgen receptor and synergise with enzalutamide.

Prostate cancer is the second most frequent cancer diagnosed in men, and accounts for one-fifth of cancer associated deaths worldwide. Despite the availability of effective prostate cancer therapies, if it is not cured by radical local treatment, progression to drug resistant metastatic prostate cancer is inevitable. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. We have recently published research demonstrating that targeting the endosomal recycling pathway, a membrane transport pathway that recycles internalised cell surface proteins back to the plasma membrane, may be a novel means to downregulate clinically relevant cell surface proteins and potentially overcome drug resistance. A reverse phase protein array (RPPA) assay of breast cancer cells treated with an endosomal recycling inhibitor identified the androgen receptor (AR) as one of the top downregulated proteins. We confirmed that endosomal recycling inhibitors also downregulated AR in prostate cancer cells and show that this occurs at the transcriptional level. We also found that endosomal recycling inhibitors synergise with enzalutamide, a standard-of-care therapy for prostate cancer. Our data suggest that combining recycling inhibitors with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

The development of HER2-targeted therapies has led to a dramatic improvement in outcomes for breast cancer patients. However, nearly all patients with metastatic HER2-positive breast cancer will eventually progress on these therapies due to innate or acquired resistance. Recent evidence suggests that the endosomal recycling of HER2 plays an important role in regulating its oncogenic signalling. Here we report that the expression of Rab coupling protein (RCP), a key regulator of endosomal recycling, positively correlates with that of HER2 and HER3 in breast tumours, and high RCP expression is predictive of poor relapse-free and overall survival in patients with HER2-amplified breast cancer. Chemical and genetic inhibition of endosomal recycling leads to a reduction in the total cellular levels of HER2 and HER3 and inhibits the activation of their downstream signalling pathways. We find that HER2 and HER3 that have been internalised from the plasma membrane are diverted to lysosomes for degradation when endosomal recycling is blocked. Primaquine (PQ), a small molecule inhibitor of the endosomal recycling pathway, synergises with HER2-targeting tyrosine kinase inhibitors and overcomes innate and acquired resistance to these TKIs. Moreover, TKI-induced drug tolerant persister cells are vulnerable to endosomal recycling inhibitors. These findings suggest that inhibition of endosomal recycling represents a promising therapeutic strategy for treating drug resistant HER2-positive breast cancer.

Ryanodine receptor calcium release channels in trophoblasts and their role in cell migration.

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca2+ ([Ca2+]c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca2+]c, whereas pharmacological inhibition of these channels reduced Ca2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.

Clinicopathological characteristics and clinical morbidity in high-risk head and neck cutaneous squamous cell carcinoma patients in Western Australia.

BACKGROUND: There is no registry data on morbidity and mortality of high-risk cutaneous squamous cell carcinoma (cSCC) in Australia. AIM: To examine the clinicopathological features, mortality and morbidity in high-risk cSCC patients in Western Australia (WA). METHODS: A retrospective cohort study was conducted through hospital record review on cSCC patients discussed at multidisciplinary meetings at the two largest WA hospitals between March 2015 and December 2016. RESULTS: Of 141 patients, 129 were evaluable, with median follow up of 43.9 (range 3.0-53.2) months. Patients were predominantly older males (84%) with significant comorbidities (Charlson Comorbidity Index (CCI) ≥5; 76%) and history of previous nonmelanoma skin cancer (57%) with advanced disease (57% stage IV without distant metastasis; American Joint Committee on Cancer, 7th edition). Pathological high-risk features were common including nodal extracapsular extension (47%) and cranial nerve involvement (16%). Clinical morbidity was significant with a median of 2 (range 0-13) excisions and 2 (range 0-21) cSCC-related hospitalisations for any cSCC event following the index case discussion. Recurrences of the primary index lesion occurred in 60% of patients and 20% had ≥2 recurrences. Median overall survival for patients with nonmetastatic disease was 39.8 (range 25.9-53.7) months and 16.1 (range 0.2-32.0) months for metastatic disease. CCI ≥5, advanced nodal stage and ≥2 recurrences were significantly associated with mortality on multivariable analyses (P < 0.05). Nodal extracapsular extension and any recurrences were identified as significant risk factors for disease-specific mortality on multivariable analyses (P < 0.05). CONCLUSION: High-risk cSCC patients have significant health needs represented by high-baseline comorbidities, multiplicity of cSCC events and the number of healthcare-associated interventions. There is an unmet need for robust cancer data collection.

The Endosomal Recycling Pathway-At the Crossroads of the Cell.

The endosomal recycling pathway lies at the heart of the membrane trafficking machinery in the cell. It plays a central role in determining the composition of the plasma membrane and is thus critical for normal cellular homeostasis. However, defective endosomal recycling has been linked to a wide range of diseases, including cancer and some of the most common neurological disorders. It is also frequently subverted by many diverse human pathogens in order to successfully infect cells. Despite its importance, endosomal recycling remains relatively understudied in comparison to the endocytic and secretory transport pathways. A greater understanding of the molecular mechanisms that support transport through the endosomal recycling pathway will provide deeper insights into the pathophysiology of disease and will likely identify new approaches for their detection and treatment. This review will provide an overview of the normal physiological role of the endosomal recycling pathway, describe the consequences when it malfunctions, and discuss potential strategies for modulating its activity.

A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma.

PURPOSE: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. EXPERIMENTAL DESIGN: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. RESULTS: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. CONCLUSION: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

Circulating microRNAs as promising diagnostic biomarkers for pancreatic cancer: a systematic review.

Pancreatic cancer (PC) is one of the most common forms of malignant tumors and causes of tumor-related death worldwide. The current prognosis of PC still remains poor due to the lack of effective early detection method. Recently, there is strong support that circulating miRNAs can be used as biomarkers for early detection of various cancers, including PC. The purpose of this review is to provide an overview of previous published studies on circulating miRNAs in plasma/serum for early detection of PC and summarize their diagnostic value. PubMed, Embase and Web of Science were systematically searched for eligible studies on circulating miRNAs for PC detection. Overall, 29 studies published between 2009 and 2018 evaluating 51 individual miRNAs (no P-value exceeding 0.05) and 13 miRNAs panels were included. Generally, the diagnostic performance of circulating miRNAs for PC detection was strong, with both the sensitivity and specificity of 36% individual miRNAs and 40% miRNAs panels exceeding 80%. Moreover, two promising miRNA panels were discovered and verified externally with all AUC values exceeding 0.95. Therefore, circulating miRNAs may hold potential to be used as noninvasive diagnostic biomarkers for PC, but large-scale studies are still needed to validate the promising miRNAs and optimize the miRNA panels. Since, the tremendous heterogeneity of studies in this field hampers translating miRNA markers into clinical practice, miRNA analytical procedures are also needed to be standardized in the future.

The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3.

Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1 This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD.This article has an associated First Person interview with the first author of the paper.

Pre-treatment wait time for head and neck cancer patients in Western Australia: description of a new metric and examination of predictive factors.

BACKGROUND: Prolonged pre-treatment wait times in head and neck cancer are associated with increased morbidity and reduced survival. Traditional metrics exclude delays prior to biopsy, which represents an important and measurable period of time. This study aims to describe total wait time for head and neck cancer patients in our institution, to define a more accurate representation of the clinically relevant pre-treatment wait time, and to evaluate predictive factors for prolonged wait times. METHODS: A retrospective review of head and neck cancer patients treated over 2 years in a tertiary referral centre was conducted. Patient demographics, referral symptoms, tumour details, treatment plan and key dates were analysed to identify total wait time and factors predictive of increased wait time. RESULTS: Two hundred and ninety-four patients were included. Mean total wait time from initial referral to treatment initiation was 71.6 (median 61) days. The period from referral to biopsy represented 29% of mean total wait time. Factors predictive of increased wait time included presenting symptom of hoarseness, laryngeal cancer and treatment with definitive radiotherapy. CONCLUSIONS: This study demonstrates that time from referral to biopsy represents a significant portion of total wait time, and we suggest that this be incorporated into future wait time metrics for improved clinical relevance. Furthermore, we have identified factors predicting increased wait time which can be targeted for future service improvement.

Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5ß1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

Mitigating the heroin crisis in Baltimore, MD, USA: a cost-benefit analysis of a hypothetical supervised injection facility.

BACKGROUND: In Baltimore, MD, as in many cities throughout the USA, overdose rates are on the rise due to both the increase of prescription opioid abuse and that of fentanyl and other synthetic opioids in the drug market. Supervised injection facilities (SIFs) are a widely implemented public health intervention throughout the world, with 97 existing in 11 countries worldwide. Research has documented the public health, social, and economic benefits of SIFs, yet none exist in the USA. The purpose of this study is to model the health and financial costs and benefits of a hypothetical SIF in Baltimore. METHODS: We estimate the benefits by utilizing local health data and data on the impact of existing SIFs in models for six outcomes: prevented human immunodeficiency virus transmission, Hepatitis C virus transmission, skin and soft-tissue infection, overdose mortality, and overdose-related medical care and increased medication-assisted treatment for opioid dependence. RESULTS: We predict that for an annual cost of $1.8 million, a single SIF would generate $7.8 million in savings, preventing 3.7 HIV infections, 21 Hepatitis C infections, 374 days in the hospital for skin and soft-tissue infection, 5.9 overdose deaths, 108 overdose-related ambulance calls, 78 emergency room visits, and 27 hospitalizations, while bringing 121 additional people into treatment. CONCLUSIONS: We conclude that a SIF would be both extremely cost-effective and a significant public health and economic benefit to Baltimore City.

Regulation of NF-κB by PML and PML-RARα.

Promyelocytic Leukemia (PML) is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. PML is a tumour suppressor and regulator of cell differentiation. We demonstrate that PML promotes TNFα-induced transcriptional responses by promoting NF-κB activity. TNFα-treated PML-/- cells show normal IκBα degradation and NF-κB nuclear translocation but significantly reduced NF-κB DNA binding and phosphorylation of NF-κB p65. We also demonstrate that the PML retinoic acid receptor-α (PML-RARα) oncofusion protein, which causes acute promyelocytic leukemia, inhibits TNFα induced gene expression and phosphorylation of NF-κB. This study establishes PML as an important regulator of NF-κB and demonstrates that PML-RARα dysregulates NF-κB.

Reconstruction of maxillary defect with musculo-adipose rectus free flap.

BACKGROUND: The rectus myocutaneous free flap (RMFF) is used for medium to large maxillectomy defects. However, in patients with central obesity the inset could be difficult due to the bulk from excessive layer of adipose tissue. We describe a modification of the RMFF for patients with excessive central obesity with a flap consisting of adipose tissue with minimal rectus muscle; the musculo-adipose rectus free flap (MARF). METHODS: Five cases of MARF reconstruction were performed between 2003 and 2013, with patients' body mass indexes ranging from 29.0 to 41.2 kg/m2 . All patients had sinonasal tumor, of which three were adenoid cystic carcinoma, one squamous cell carcinoma, and one melanoma. Four patients had Codeiro IIIb defects and one had Codeiro II defect. Using the MARF technique, the maxillectomy defect was obliterated with vascularized adipose tissue overlying the rectus muscle and was trimmed to fit the maxillectomy defect. The adipose tissue was allowed to granulate and mucosalize. RESULTS: The volume of adipose tissue harvested was between 120 and 160 mL. All flaps survived with no requirement for re-exploration. Complete oro-nasal separation was achieved in all patients. The time to commencement of oral intake ranges from 5 to 15 days. One patient developed seroma and one developed wound breakdown on the donor site. The length of stay at the hospital ranges from 9 to 22 days. On follow-up ranging 7.5-32.8 months, two patients died from their malignancies. The other three patients were able to tolerate oral soft diet. CONCLUSION: The MARF may be considered as an alternative to myocutaneous rectus free flap particularly for the reconstruction of maxillary defects in patients with central obesity. © 2015 Wiley Periodicals, Inc. Microsurgery 37:137-141, 2017.

Myosin Va is required for the transport of fragile X mental retardation protein (FMRP) granules.

BACKGROUND INFORMATION: Fragile X mental retardation protein (FMRP) is a selective RNA binding protein that functions as a translational inhibitor. It also plays a role in directing the transport of a subset of mRNAs to their site of translation and several recent reports have implicated microtubule motor proteins in the transport of FMRP-messenger ribonucleoprotein (mRNP) granules in neurons. Earlier work reported the association of the actin-based motor protein myosin Va with FMRP granules. RESULTS: Here, we follow up on this finding and confirm that myosin Va does in fact associate with FMRP and is required for its correct intracellular localisation. FMRP is concentrated in the perinuclear region of myosin Va-null mouse melanoma cells which contrasts starkly with the evenly distributed punctate pattern observed in wild-type cells. Similarly, overexpression of a dominant-negative mutant of myosin Va results in the accumulation of FMRP in large aggregate-like structures. FRAP experiments demonstrate that FMRP is largely immobile in the absence of myosin Va. CONCLUSIONS: Combining these data, we propose a model in which myosin Va and kinesin play key roles in the assembly and subsequent transport of FMRP granules along microtubules to the periphery of the cell. Myosin Va captures the complex onto peripheral actin structures and mediates the local delivery of the FMRP granule to the site of mRNA translation.