Amanda Harvey

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts

BackgroundRadial chromosome positioning in interphase nuclei is nonrandom and can alter according to developmental, differentiation, proliferation, or disease status. However, it is not yet clear when and how chromosome repositioning is elicited.ResultsBy investigating the positioning of all human chromosomes in primary fibroblasts that have left the proliferative cell cycle, we have demonstrated that in cells made quiescent by reversible growth arrest, chromosome positioning is altered considerably. We found that with the removal of serum from the culture medium, chromosome repositioning took less than 15 minutes, required energy and was inhibited by drugs affecting the polymerization of myosin and actin. We also observed that when cells became quiescent, the nuclear distribution of nuclear myosin 1β was dramatically different from that in proliferating cells. If we suppressed the expression of nuclear myosin 1β by using RNA-interference procedures, the movement of chromosomes after 15 minutes in low serum was inhibited. When high serum was restored to the serum-starved cultures, chromosome repositioning was evident only after 24 to 36 hours, and this coincided with a return to a proliferating distribution of nuclear myosin 1β.ConclusionsThese findings demonstrate that genome organization in interphase nuclei is altered considerably when cells leave the proliferative cell cycle and that repositioning of chromosomes relies on efficient functioning of an active nuclear motor complex that contains nuclear myosin 1β.

Use of RNA interference to validate Brk as a novel therapeutic target in breast cancer: Brk promotes breast carcinoma cell proliferation

Brk (PTK6) is a nonreceptor protein tyrosine kinase, which is expressed in over 60% of breast carcinoma tissue samples and breast tumour cell lines, but not normal mammary tissue or benign lesions. Since experimental Brk expression in nontransformed mammary epithelial cells enhances their mitogenic response to epidermal growth factor, it was important to determine the role Brk plays in the proliferation of breast carcinoma cells and validate it as a therapeutic target. We have used RNA interference to efficiently and specifically downregulate Brk protein levels in breast carcinoma cells, and determined that this results in a significant suppression of their proliferation. Additionally, through the expression of a kinase-inactive mutant, we have determined that Brk can mediate promotion of proliferation via a kinase-independent mechanism, potentially functioning as an ‘adapter’. These data identify Brk as a novel target for antiproliferative therapy in the majority of breast cancers, and illustrate the power of RNA interference for rapidly validating candidate therapeutic targets.

Brk Protects Breast Cancer Cells from Autophagic Cell Death Induced by Loss of Anchorage

Brk, a tyrosine kinase expressed in a majority of breast tumors, but not normal mammary tissue, promotes breast carcinoma cell proliferation. Normal epithelial cells are dependent on cell-cell or cell-matrix interactions for survival and undergo apoptosis after disruption of these interactions. Tumor cells are less sensitive to the induction of apoptosis and are predicted to have the potential to disseminate. We investigated whether Brk has further roles in breast tumor progression by relating its expression to tumor grade and demonstrating its role in the regulation of carcinoma cell survival under non-adherent conditions. Brk expression was determined by reverse transcription PCR on RNA extracted from surgical samples of human breast cancers. Breast carcinoma cell survival in suspension culture was examined when Brk protein levels were suppressed by RNA interference. Additionally, the effect of experimentally overexpressing Brk in otherwise Brk-negative breast carcinoma cells was assessed. Brk mRNA expression was notably higher in grade 3 breast tumors, as compared with lower tumor grades. In suspension culture, Brk suppression increased the rate of cell death, as compared with controls, and this cell death program exhibited characteristics of autophagy but not of apoptosis. Conversely, experimental expression of Brk in Brk-negative cells increased cell survival whereas kinase-inactive Brk did not. Therefore, Brk enhances breast carcinoma cell survival in suspension, suggesting a role for Brk in supporting breast cancer cell dissemination.

Targeting primary human leukaemia cells with RNA interference: Bcr-Abl targeting inhibits myeloid progenitor self- renewal in chronic myeloid leukaemia cells

We have investigated functional outcome of challenging primary chronic myeloid leukaemia (CML) cells with Bcr‐Abl fusion sequence‐directed RNA interference (RNAi). We targeted the Bcr‐Abl b3a2 variant, by RNAi, in primary chronic phase CML cells, and detected strikingly reduced proliferation of myeloid precursor cells expressing this variant. Lack of an effect in cells expressing a distinct Bcr‐Abl variant confirmed the specificity of the response. Through the functional targeting of an oncogene in primary human tumour cells, we have demonstrated that Bcr‐Abl enhances CML progenitor cell amplification, and that RNAi may be suitable for development as a specific anti‐leukaemia treatment.

The Brk protein tyrosine kinase as a therapeutic target in cancer: opportunities and challenges.

Brk is an intracellular protein tyrosine kinase that is significantly overexpressed in a majority of breast tumors, while being detected at appreciable levels in only a limited range of adult tissues that does not include the mammary gland. It has recently been demonstrated to have a role in promoting the proliferation of carcinoma cells, one that it is unlikely to perform in normal adult cells, and it therefore represents an exciting target for the development of novel cancer therapies based on specifically or selectively interfering with its functions. The strategy of pharmaceutical kinase inhibition is clinically proven and widely pursued in oncology programmes directed at a variety of tumor types. However, a potentially kinase-independent role for Brk in regulating proliferation suggests that alternative approaches, such as inhibiting protein–protein interactions, may prove more successful. Further research into Brks signaling functions will underpin progress towards turning the potential suggested by these observations into rational drug discovery, from which a large number of patients stand to benefit.

The clinicopathological significance of lamin A/C, lamin B1 and lamin B receptor mRNA expression in human breast cancer

Lamin A/C (LMNA), lamin B1 (LMNB1) and lamin B receptor (LBR) have key roles in nuclear structural integrity and chromosomal stability. In this study, we have studied the relationships between the mRNA expressions of A-type lamins, LMNB1 and LBR and the clinicopathological parameters in human breast cancer. Samples of breast cancer tissues (n = 115) and associated non-cancerous tissue (ANCT; n = 30) were assessed using reverse transcription and quantitative PCR. Transcript levels were correlated with clinicopathological data. Higher levels of A-type lamins and LMNB1 mRNA expression were seen in ANCT. Higher lamin A/C expression was associated with the early clinical stage (TNM1 vs. TNM3 — 13 vs. 0.21; p = 0.0515), with better clinical outcomes (disease-free survival vs. mortality — 11 vs. 1; p = 0.0326), and with better overall (p = 0.004) and disease-free survival (p = 0.062). The expression of LMNB1 declined with worsening clinical outcome (disease-free vs. mortalities — 0.0011 vs. 0.000; p = 0.0177). LBR mRNA expression was directly associated with tumor grade (grade 1 vs. grade 3 — 0.00 vs. 0.00; p = 0.0479) and Nottingham Prognostic Index (NPI1 vs. NPI3 — 0.00 vs. 0.00; p = 0.0551). To the best of our knowledge, this is the first study to suggest such a role for A-type lamins, lamin B1 and LBR in human breast cancer, identifying an important area for further research.

HIF-1α-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion

PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease.

Opposing actions of TGFbeta1 and FGF2 on growth, differentiation and extracellular matrix accumulation in prostatic stromal cells.

TGFβ1 and FGF2 are autocrine growth factors in prostatic stroma and are elevated in benign prostatic hyperplasia (BPH), a disease characterized by enlargement of the stromal compartment of the prostate. TGFβ1 has a biphasic effect on proliferation of prostatic stromal cells, inducing proliferation at low doses ( < 1 ng/ml), but inhibiting growth above 1 ng/ml. This study investigated the role of TGFβ1 and FGF2 on growth factor bioavailability and extracellular matrix (ECM) accumulation synthesis in cultured prostatic stromal cells. Real-Time-PCR showed that TGFβ1 expression is auto-inductive, whereas FGF2 is auto-repressive. FGF2 also induced TGFβ1 secretion in the absence of increased TGFβ1 mRNA expression. TGFβ1 and FGF2 have opposing actions on Type 1 collagen expression, a finding confirmed by Western blotting. The bioavailability of TGFβ1 regulated by FGF2 may represent part of a negative feedback mechanism controlling stromal growth, differentiation and ECM. Dysregulation of this pathway in favour of TGFβ1 bioactivity may exacerbate BPH.

Breast tumor kinase (Brk/PTK6) plays a role in the differentiation of primary keratinocytes

Breast Tumor Kinase (Brk/PTK6) has a relatively limited expression profile in normal tissue. Its expression is restricted to epithelial cells that are differentiating such as those in the epidermis, and Brk expression appears to be absent from proliferating cells in normal tissue. Also, there is now some evidence to suggest that Brk plays a functional role in the differentiation of the keratinocytes in the epidermis. We have, therefore, investigated the role that Brk/PTK6 plays in normal human primary keratinocytes by suppressing protein levels using RNA interference. We show that as primary human keratinocytes are induced to differentiate in vitro, Brk levels decrease. Decreasing Brk protein levels lead to an increase in the number of cells with a permeable plasma membrane, a decrease in epidermal growth factor receptor (EGFR) and a parallel increase in keratin 10 levels, but classical markers of apoptosis or terminal differentiation are not affected. We propose Brk, Keratin 10 and EGFR are co-regulated during differentiation and that manipulating Brk expression can influence the differentiation of normal primary human keratinocytes.