Resistance to HER2-targeted anti-cancer drugs is associated with immune evasion in cancer cells and their derived extracellular vesicles.

Neuromedin U (NmU) -a neuropeptide belonging to the neuromedin family- plays a substantial role in HER2-positive breast cancer, correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients. However, the mechanism through which it exerts these effects remains unclear. To elucidate this, initially we used HER2-positive breast cancer cells stably over-expressing NmU. These cells and their released extracellular vesicles (EVs) had increased amounts of the immunosuppressive cytokine TGFß1 and the lymphocyte activation inhibitor PD-L1. Furthermore, these cells also showed enhanced resistance to antibody-dependent cell cytotoxicity (ADCC) mediated by trastuzumab, indicating a role of NmU in enhancing immune evasion. All these features were also found in HER2-targeted drug-resistant cells which we previously found to express higher levels of NmU than their drug-sensitive counterparts. Interestingly, EVs from drug-resistant cells were able to increase levels of TGFß1 in drug-sensitive cells. In our neo-adjuvant clinical trial, TGFß1 levels were significantly higher in EVs isolated from the serum of patients with HER2-overexpressing breast cancers who went on to not respond to HER2-targeted drug treatment, compared with those who experienced complete or partial response. Taken together, our results report a new mechanism-of-action for NmU in HER2-overexpressing breast cancer that enhances resistance to the anti-tumor immune response. Furthermore, EV levels of TGFß1 correlating with patients' response versus resistance to HER2-targeted drugs suggests a potential use of EV-TGFß1 as a minimally-invasive companion diagnostic for such treatment in breast cancer.

Neratinib resistance and cross-resistance to other HER2-targeted drugs due to increased activity of metabolism enzyme cytochrome P4503A4.

BACKGROUND: Neratinib is in Phase 3 clinical trials but, unfortunately, the development of resistance is inevitable. Here, we investigated the effects of acquired neratinib resistance on cellular phenotype and the potential mechanism of this resistance. METHODS: Neratinib-resistant variants of HER2-positive breast cancer cells were developed and their cross-resistance investigated using cytotoxicity assays. Similarly, sensitivity of trastuzumab-resistant and lapatinib-resistant cells to neratinib was assessed. Cellular phenotype changes were evaluated using migration, invasion and anoikis assays. Immunoblotting for HER family members and drug efflux pumps, as well as enzyme activity assays were performed. RESULTS: Neratinib resistance conferred cross-resistance to trastuzumab, lapatinib and afatinib. Furthermore, the efficacy of neratinib was reduced in trastuzumab- and lapatinib-resistant cells. Neratinib-resistant cells were more aggressive than their drug-sensitive counterparts, with increased CYP3A4 activity identified as a novel mechanism of neratinib resistance. CONCLUSIONS: The potential of increased CYP3A4 activity as a biomarker and/or target to add value to neratinib warrants investigation.

The relevance of using 3D cell cultures, in addition to 2D monolayer cultures, when evaluating breast cancer drug sensitivity and resistance.

Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.

Receptor tyrosine kinase targeting in multicellular spheroids.

While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids.

Neuromedin U: a candidate biomarker and therapeutic target to predict and overcome resistance to HER-tyrosine kinase inhibitors.

Intrinsic and acquired resistance to HER-targeting drugs occurs in a significant proportion of HER2-overexpressing breast cancers. Thus, there remains a need to identify predictive biomarkers that could improve patient selection and circumvent these types of drug resistance. Here, we report the identification of neuromedin U (NmU) as an extracellular biomarker in cells resistant to HER-targeted drugs. NmU overexpression occurred in cells with acquired or innate resistance to lapatinib, trastuzumab, neratinib, and afatinib, all of which displayed a similar trend upon short-term exposure, suggesting NmU induction may be an early response. An analysis of 3,489 cases of breast cancer showed NmU to be associated with poor patient outcome, particularly those with HER2-overexpressing tumors independent of established prognostic indicators. Ectopic overexpression of NmU in drug-sensitive cells conferred resistance to all HER-targeting drugs, whereas RNAi-mediated attenuation sensitized cells exhibiting acquired or innate drug resistance. Mechanistic investigations suggested that NmU acted through HSP27 as partner protein to stabilize HER2 protein levels. We also obtained evidence of functional NmU receptors on HER2-overexpressing cells, with the addition of exogenous NmU eliciting an elevation in HER2 and EGFR expression along with drug resistance. Finally, we found that NmU seemed to function in cell motility, invasion, and anoikis resistance. In vivo studies revealed that NmU attenuation impaired tumor growth and metastasis. Taken together, our results defined NmU as a candidate drug response biomarker for HER2-overexpressing cancers and as a candidate therapeutic target to limit metastatic progression and improve the efficacy of HER-targeted drugs.

miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer.

BACKGROUND: While the treatment of HER2 over-expressing breast cancer with recent HER-targeted drugs has been highly effective for some patients, primary (also known as innate) or acquired resistance limits the success of these drugs. microRNAs have potential as diagnostic, prognostic and predictive biomarkers, as well as replacement therapies. Here we investigated the role of microRNA-630 (miR-630) in breast cancer progression and as a predictive biomarker for response to HER-targeting drugs, ultimately yielding potential as a therapeutic approach to add value to these drugs. METHODS: We investigated the levels of intra- and extracellular miR-630 in cells and conditioned media from breast cancer cell lines with either innate- or acquired- resistance to HER-targeting lapatinib and neratinib, compared to their corresponding drug sensitive cell lines, using qPCR. To support the role of miR-630 in breast cancer, we examined the clinical relevance of this miRNA in breast cancer tumours versus matched peritumours. Transfection of miR-630 mimics and inhibitors was used to manipulate the expression of miR-630 to assess effects on response to HER-targeting drugs (lapatinib, neratinib and afatinib). Other phenotypic changes associated with cellular aggressiveness were evaluated by motility, invasion and anoikis assays. TargetScan prediction software, qPCR, immunoblotting and ELISAs, were used to assess miR-630's regulation of mRNA, proteins and their phosphorylated forms. RESULTS: We established that introducing miR-630 into cells with innate- or acquired- resistance to HER-drugs significantly restored the efficacy of lapatinib, neratinib and afatinib; through a mechanism which we have determined to, at least partly, involve miR-630's regulation of IGF1R. Conversely, we demonstrated that blocking miR-630 induced resistance/insensitivity to these drugs. Cellular motility, invasion, and anoikis were also observed as significantly altered by miR-630 manipulation, whereby introducing miR-630 into cells reduced cellular aggression while inhibition of miR-630 induced a more aggressive cellular phenotype. CONCLUSIONS: Taken together, our findings suggest miR-630 as a key regulator of cancer cell progression in HER2 over-expressing breast cancer, through targeting of IGF1R. This study supports miR-630 as a diagnostic and a predictive biomarker for response to HER-targeted drugs and indicates that the therapeutic addition of miR-630 may enhance and improve patients' response to HER-targeting drugs.

Deployment of lean six sigma in care coordination: an improved discharge process.

PURPOSE/OBJECTIVES: This article presents a quality improvement project to reduce readmissions in the Medicare population related to heart failure, acute myocardial infarction, and pneumonia. The article describes a systematic approach to the discharge process aimed at improving transitions of care from hospital to post-acute care, utilizing Lean Six Sigma methodology. PRIMARY PRACTICE SETTING: Inpatient acute care hospital. FINDINGS/CONCLUSIONS: A coordinated discharge process, which includes postdischarge follow-up, can reduce avoidable readmissions. Implications for CASE MANAGEMENT: The quality improvement project demonstrated the significant role case management plays in preventing costly readmissions and improving outcomes for patients through better transitions of care from the hospital to the community. By utilizing Lean Six Sigma methodology, hospitals can focus on eliminating waste in their current processes and build more sustainable improvements to deliver a safe, quality, discharge process for their patients. Case managers are leading this effort to improve care transitions and assure a smoother transition into the community postdischarge..

Three-dimensional cell culture: the missing link in drug discovery.

Cells, grown as monolayers (2D models), are routinely used as initial model systems for evaluating the effectiveness and safety of libraries of molecules with potential as therapeutic drugs. While this initial screening precedes preclinical animal studies before advancing to human clinical trials, cultured cells frequently determine the initial, yet crucial, 'stop/go' decisions on the progressing of the development of a drug. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo, for example, akin to a tumour, and possibly co-cultured with other cells and cellular components that naturally occur in their microenvironment may be more clinically relevant. Here, in the context of anti-cancer drug screening, we review 2D and 3D culture approaches, consider the strengths and relevance of each method.

Wireless technology improves nursing workflow and communications.

Inpatient healthcare delivery involves complex processes that require interdisciplinary teamwork and frequent communication among physicians, nurses, unit secretaries, and ancillary staff. Often, these interactions are not at a nursing unit, or near a phone. In an effort to address the inefficiencies of these workflow processes and communications, St. Agnes HealthCare, Baltimore, MD, installed a new hands-free communications system that uses a wireless network, voice recognition, and a small wearable badge. Developed by Vocera, the communications system permits one-button access to others on the system or connects to outside phones through PBX integration. While many agree that today's technology has the potential to positively impact nursing care delivery, St. Agnes HealthCare and Vocera, with assistance from First Consulting Group, decided to conduct a comprehensive benefits study in December 2003 to quantify the impact of this communications system on workflow and communications. The results identified a number of significant findings that demonstrate its value from a quantitative and qualitative standpoint. The following article describes this study and its findings.