Evolution of breast cancer therapeutics: Breast tumour kinase’s role in breast cancer and hope for breast tumour kinase targeted therapy

There have been significant improvements in the detection and treatment of breast cancer in recent decades. However, there is still a need to develop more effective therapeutic techniques that are patient specific with reduced toxicity leading to further increases in patients’ overall survival; the ongoing progress in understanding recurrence, resistant and spread also needs to be maintained. Better understanding of breast cancer pathology, molecular biology and progression as well as identification of some of the underlying factors involved in breast cancer tumourgenesis and metastasis has led to the identification of novel therapeutic targets. Over a number of years interest has risen in breast tumour kinase (Brk) also known as protein tyrosine kinase 6; the research field has grown and Brk has been described as a desirable therapeutic target in relation to tyrosine kinase inhibition as well as disruption of its kinase independent activity. This review will outline the current “state of play” with respect to targeted therapy for breast cancer, as well as discussing Brk’s role in the processes underlying tumour development and metastasis and its potential as a therapeutic target in breast cancer.     

2nd Gordon Hamilton Fairley Lecture: Need for new approaches to the treatment of patients in clinical remission, with special reference to acute myeloid leukaemia

A serious limitation of chemotherapy for acute myeloid leukaemia (AML), Hodgkins disease and some classes of breast cancer is that, even when clinically evident disease responds well, the same chemotherapy when given during remission does not affect the rate of relapse after chemotherapeutic or surgical ablation of the primary disease. This cannot, in general, be caused by genetic adaptation of the residual cancer cells which renders them resistant to specific drugs, because after relapse further remissions can be obtained with the same drugs that were ineffective by chronic administration in prolonging remission. The resistance of the residual cells may arise from mechanisms such as inaccessibility for anatomical or other reasons, or because of a change in metabolic state which causes these cells temporarily to cease division, when they cannot be harmed by cycle-dependent drugs and repair damage sustained from cycle-independent drugs. Limited differentiation has been shown capable of reversal and this may be a mechanism which leads to quiescence and associated “resistance”, particularly in the case of AML. Where such resistance occurs treatment during remission—or as an adjuvant to surgery and radiotherapy—may have to rely on mechanisms which are independent of cellular proliferation such as processes associated with graft-versus-host-disease or the induction of terminal differentiation. A model for studying the nature of resistance of residual cancer and for testing treatments that might be active against cancer cells in this state may be dormant metastases. The latter are malignant cells which appear to be in peaceful co-existence with their host and which in experimental systems have been induced to grow into lethal metastases by perturbation of the host by surgical trauma, by hormonal manipulation or by immunosuppression.     

Understanding the roles of N6-methyladenosine writers,readers and erasers in breast cancer

Breast cancer is believed to be driven by epigenetic regulation of genes implicated in cell proliferation, survival, and differentiation. Recently, aberrant N⁶-methyladenosine (m⁶A) decorations turned up as crucial epigenetic regulator for malignant breast cancer, which may serve as new targets for breast cancer treatment. Here we briefly outline the functions of m⁶A and its regulatory proteins, including m⁶A “writers,” “readers,” and “erasers” on RNA life fate, recapitulate the latest breakthroughs in understanding m⁶A modification and its regulatory proteins, and the underlying molecular mechanisms that contribute to the carcinogenesis and the progression of breast cancer, so as to provide potential epigenetic targets for diagnosis, treatment and prognosis in breast cancer.     

Cancer Metabolism: New Validated Targets for Drug Discovery

Recent studies in cancer metabolism directly implicate catabolic fibroblasts as a new rich source of i) energy and ii) biomass, for the growth and survival of anabolic cancer cells. Conversely, anabolic cancer cells upregulate oxidative mitochondrial metabolism, to take advantage of the abundant fibroblast fuel supply. This simple model of “metabolic-symbiosis” has now been independently validated in several different types of human cancers, including breast, ovarian, and prostate tumors. Biomarkers of metabolic-symbiosis are excellent predictors of tumor recurrence, metastasis, and drug resistance, as well as poor patient survival. New pre-clinical models of metabolic-symbiosis have been generated and they genetically validate that catabolic fibroblasts promote tumor growth and metastasis. Over 30 different stable lines of catabolic fibroblasts and >10 different lines of anabolic cancer cells have been created and are well-characterized. For example, catabolic fibroblasts harboring ATG16L1 increase tumor cell metastasis by >11.5-fold, despite the fact that genetically identical cancer cells were used. Taken together, these studies provide >40 novel validated targets, for new drug discovery and anti-cancer therapy. Since anabolic cancer cells amplify their capacity for oxidative mitochondrial metabolism, we should consider therapeutically targeting mitochondrial biogenesis and OXPHOS in epithelial cancer cells. As metabolic-symbiosis promotes drug-resistance and may represent the escape mechanism during anti-angiogenic therapy, new drugs targeting metabolic-symbiosis may also be effective in cancer patients with recurrent and advanced metastatic disease.     

A class of genes in the HER2 regulon that is poised for transcription in breast cancer cell lines and expressed in human breast tumors

HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2⁺ and HER2⁻ breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/− breast cancer tissues. Genes differentially expressed between HER2+/− cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2⁺ cell lines, but without significant gene expression. Of 737 such genes “poised” for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A “poised” class of genes in HER2⁺ cell lines with POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue.     

p70S6 kinase mediates breast cancer cell survival in response to surgical wound fluid stimulation

In early breast cancer, local relapses represent a determinant and not simply an indicator of risk for distant relapse and death. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Relevance of PI3K/mTOR/p70S6K signaling in breast tumorigenesis is very well documented. However, the pathway/s involved in the process of breast cancer local relapse are not well understood. The ribosomal protein p70S6K has been implicated in breast cancer cell response to post‐surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. Here, we show that p70S6K activity is required for the survival of breast cancer cells challenged in “hostile” microenvironments. We found that impairment of p70S6K activity in breast cancer cells strongly decreased their tumor take rate in nude mice. In line with this observation, if cells were challenged to grow in anchorage independence or in clonogenic assay, growth of colonies was strongly dependent on an intact p70S6K signaling. This in vitro finding was particularly evident when breast cancer cells were grown in the presence of wound fluids harvested following surgery from breast cancer patients, suggesting that the stimuli present in the post‐surgical setting at least partially relied on activity of p70S6K to stimulate breast cancer relapse. From a mechanistic point of view, our results indicated that p70S6K signaling was able to activate Gli1 and up‐regulate the anti‐apoptotic protein Bcl2, thereby activating a survival response in breast cancer cells challenged in hostile settings. Our work highlights a previously poorly recognized function of p70S6K in preserving breast cancer cell survival, which could eventually be responsible for local relapse and opens the way to the design of new and more specific therapies aiming to restrain the deleterious effects of wound response.     

Monocytes and macrophages,implications for breast cancer migration and stem cell-like activity and treatment

Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with poor prognosis and high grade tumours we used the THP-1 cell line to model monocyte-macrophage differentiation in co-culture with four breast cancer cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468) to model in vivo cellular interactions. Polarisation into M1 and M2 subtypes was confirmed by specific cell marker expression of ROS and HLA-DR, respectively. Co-culture with all types of macrophage increased migration of ER-positive breast cancer cell lines, while M2-macrophages increased mammosphere formation, compared to M1-macrophages, in all breast cancer cells lines. Treatment of cells with Zoledronate in co-culture reduced the “pro-tumourigenic” effects (increased mammospheres/migration) exerted by macrophages. Direct treatment of breast cancer cells in homotypic culture was unable to reduce migration or mammosphere formation.Macrophages promote “pro-tumourigenic” cellular characteristics of breast cancer cell migration and stem cell activity. Zoledronate targets macrophages within the microenvironment which in turn, reduces the “pro-tumourigenic” characteristics of breast cancer cells. Zoledronate offers an exciting new treatment strategy for both primary and metastatic breast cancer.     

Immunotherapy and immunobiomarker in breast cancer: current practice and future perspectives

Among the new cancer cases and resulting deaths among women worldwide, breast cancer is the most significant threat to women’s health. In recent years, immunotherapy was initially used to treat patients with metastatic breast cancer, where it demonstrated its unique value by providing a novel way to improve therapeutic effects and prolong survival time. With the development of clinical trials related to immunotherapy for breast cancer, tumour vaccines, such as DNA vaccines, have been observed to improve the disease-free survival (DFS) and overall survival (OS) of patients. Monoclonal antibodies have also shown good efficacy, and adoptive cell therapies, such as CAR-T, exhibit strong tumour killing ability and good safety, and thus, these therapies may comprise a new strategy for the treatment of breast cancer. These breakthrough successes have promoted the achievement of “individualized” breast cancer treatment. Moreover, a recent study showed that patients with various cancer types with a higher tumour mutational burden (TMB) are more likely to benefit from immunotherapy. As research progresses, TMB may also demonstrate a certain clinical significance in the treatment of breast cancer. This paper reviews the latest research progress on breast cancer immunotherapy and the predictive value and application status of TMB in immunotherapy regimens for breast cancer patients to provide a reference for further in-depth studies of breast cancer immunotherapy.     

Plasma Aromatase Activity Index,Gonadotropins and Estrone Are Associated with Frailty Syndrome in Post-Menopausal Women with Breast Cancer

Frailty syndrome is associated with poor outcomes, morbidity and premature mortality. We performed a cross-sectional study to evaluate the presence of frailty syndrome based on Fried’s frailty phenotype in post-menopausal women with breast cancer. We further analyzed the association between frailty syndrome with geriatric assessments and the association with the concentration of gonadotropins LH and FSH, estrogens, androgens and the aromatase activity index in the blood. We enrolled 47 post-menopausal women with localized breast cancer (mean age 66.8 ± 1.3 years (range 52–83)) prior to the starting of adjuvant endocrine therapy. Patients were identified as “non-frail” (robust) or “prefrail/frail” if they fulfilled at least one frailty criteria. In order to determine associations among variables and to control for other variables potentially affecting frailty syndrome (age, comorbidity index and previous chemotherapy treatment), we performed a logistic regression analysis. The receiver operating characteristic curve was performed to assess the sensitivity and specificity of the hormonal concentration to discriminate prefrail/frail versus non-frail individuals. Significant positive associations were observed between the severity of frailty syndrome and estrone, FSH and LH concentrations and the aromatase activity index in the blood (p < 0.05). Further research into the role of hormonal biomarkers should be evaluated in follow-up studies in order to recommend their use as suitable biomarkers of frailty syndrome in breast cancer patients.     

Resection of the primary tumor in stage IV breast cancer

Stage IV breast cancer refers to breast cancer that has already metastasized to distant regions when initially diagnosed. Treatment for stage IV is intended to “prolong survival and palliate symptoms”. Resection of a primary tumor is considered to be “effective only at alleviating chest symptoms and providing local control” in spite of the advances of imaging examination and medication for breast cancer. Molecular target and endocrine drugs are very effective and useful to tailor-make a treatment strategy according to breast cancer subtypes. Positron emission tomography-computed tomography can detect and diagnose the very small metastases and recurrences which can potentially be cured even if they are distant metastases. Recently, many retrospective studies have reported the survival benefit of surgery for breast cancer patients with metastases and some clinical trials which confirm the surgical prognostic benefit for them have started to enrol patients. The goal of treatment has to be clearly identified: increase the patient’s survival time, provide local control or perform histology to determine the cancer’s properties. The best evidence is absolutely essential to treat patients who need surgery at the right time. We need to evaluate the treatment strategy, including primary resection for stage IV breast cancer particularly, and find new evidence by prospective analysis.     

ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p≤0.001 in BT474; p≤0.01 in SKBR3) and in vivo (p≤0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p≤0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p≤0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p≤0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.     

YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

Specific overexpression in cancer cells and evidence of oncogenic functions make Survivin an attractive target in cancer tharapy. The small molecule compound YM155 has been described as the first “Survivin suppressant” but molecular mechanisms involved in its biological activity and its clinical potential remain obscure. We herein show that YM155 exerts single agent toxicity on primary breast cancer cells grown in an ex vivo assay preserving tumor microenvironment. In vitro assays indicate that YM155 more efficiently triggers cell death in breast cancer cells (including these with stem-cell like properties) than in non tumorigenic mammary cells. YM155-induced cell death is critically dependent on autophagy and NF-kB but independent of p53 and it coïncides with DNA damage an a DNA damage response in p53-proficient cells. Our results point out a crosstalk between NF-KB and autophagy controlling YM155-induced death in breast cancer cells and argue for the potential use of YM155 as a genotoxic agent in breast cancer therapy.     

Common drugs and treatments for cancer and age-related diseases: revitalizing answers to NCI's provocative questions

In 2011, The National Cancer Institute (NCI) has announced 24 provocative questions on cancer. Some of these questions have been already answered in “NCI''s provocative questions on cancer: some answers to ignite discussion” (published in Oncotarget, 2011, 2: 1352.) The questions included “Why do many cancer cells die when suddenly deprived of a protein encoded by an oncogene?” “Can we extend patient survival by using approaches that keep tumors static?” “Why are some disseminated cancers cured by chemotherapy alone?” “Can we develop methods to rapidly test interventions for cancer treatment or prevention?” “Can we use our knowledge of aging to enhance prevention or treatment of cancer?” “What is the mechanism by which some drugs commonly and chronically used for other indications protect against cancer?” “How does obesity contribute to cancer risk?” I devoted a single subchapter to each the answer. As expected, the provocative questions were very diverse and numerous. Now I choose and combine, as a single problem, only three last questions, all related to common mechanisms and treatment of age-related diseases including obesity and cancer. Can we use common existing drugs for cancer prevention and treatment? Can we use some targeted “cancer-selective” agents for other diseases and … aging itself.     

Tumour-initiating capacity is independent of epithelial–mesenchymal transition status in breast cancer cell lines

Background:

Epithelial–mesenchymal transition (EMT) and cancer stem cells (CSCs) are considered to be crucial for cancer biology. The purpose of this study was to determine whether EMT directly led to the acquisition of tumour-initiating capacity in breast cancer cell lines.

Methods:

Epithelial–mesenchymal transition was induced in five breast cancer cell lines and one normal breast cell line by EMT-related cytokine stimulation. Mesenchymal–epithelial transition (MET) was induced by stably overexpressing miR-200c in three mesenchymal-like breast cancer cell lines. Molecular expression and cell function analysis were performed to evaluate the effect of EMT or MET on tumour-initiating capacity and other biological characteristics.

Results:

The induction of EMT did not enhance tumour-initiating capacity but, instead, conferred a CD44⁺/CD24^−/low phenotype as well as cell proliferation, migration, and resistance to doxorubicin and radiation on breast cancer cell lines. Furthermore, MET did not lead to inhibition or loss of the tumour-initiating capacity in mesenchymal-like breast cancer cell lines, but it markedly attenuated other malignant properties, including proliferation, invasion, and resistance to therapy.

Conclusions:

Epithelial–mesenchymal transition does not alter tumour-initiating capacity of breast cancer cells but some other biological characteristics. Therefore, EMT and tumour-initiating capacity may not be directly linked in breast cancer cell lines.     

Factors Associated with “Survivor Identity” in Men with Breast Cancer

Cancer patients vary in their comfort with the label “survivor”. Here, we explore how comfortable males with breast cancer (BC) are about accepting the label cancer “survivor”. Separate univariate logistic regressions were performed to assess whether time since diagnosis, age, treatment status, and cancer stage were associated with comfort with the “survivor” label. Of the 70 males treated for BC who participated in the study, 58% moderately-to-strongly liked the term “survivor”, 26% were neutral, and 16% moderately-to-strongly disliked the term. Of the factors we explored, only a longer time since diagnosis was significantly associated with the men endorsing a survivor identity (OR = 1.02, p = 0.05). We discuss how our findings compare with literature reports on the comfort with the label “survivor” for women with BC and men with prostate cancer. Unlike males with prostate cancer, males with BC identify as “survivors” in line with women with BC. This suggests that survivor identity is more influenced by disease type and treatments received than with sex/gender identities.     

Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth

We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the “Reverse Warburg Effect,” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a “pseudo-starvation” response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3- hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).     

Misinformation and Facts about Breast Cancer Screening

Quality medical practice is based on science and evidence. For over a half-century, the efficacy of breast cancer screening has been challenged, particularly for women aged 40–49. As each false claim has been raised, it has been addressed and refuted based on science and evidence. Nevertheless, misinformation continues to be promoted, resulting in confusion for women and their physicians. Early detection has been proven to save lives for women aged 40–74 in randomized controlled trials of mammography screening. Observational studies, failure analyses, and incidence of death studies have provided evidence that there is a major benefit when screening is introduced to the general population. In large part due to screening, there has been an over 40% decline in deaths from breast cancer since 1990. Nevertheless, misinformation about screening continues to be promoted, adding to the confusion. Despite claims to the contrary, a careful reading of the guidelines issued by major groups such as the U.S. Preventive Services Task Force and the American College of Physicians shows that they all agree that most lives are saved by screening starting at the age of 40. There is no scientific support for using the age of 50 as a threshold for screening. All women should be provided with the facts and not false information about breast cancer screening so that they can make “informed decisions” for themselves about whether to participate.