Bone morphogenetic proteins and their antagonists: current and emerging clinical uses

Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily of secreted cysteine knot proteins that includes TGFβ₁, nodal, activins and inhibins. BMPs were first discovered by Urist in the 1960s when he showed that implantation of demineralized bone into intramuscular tissue of rabbits induced bone and cartilage formation. Since this seminal discovery, BMPs have also been shown to play key roles in several other biological processes, including limb, kidney, skin, hair and neuronal development, as well as maintaining vascular homeostasis. The multifunctional effects of BMPs make them attractive targets for the treatment of several pathologies, including bone disorders, kidney and lung fibrosis, and cancer. This review will summarize current knowledge on the BMP signalling pathway and critically evaluate the potential of recombinant BMPs as pharmacological agents for the treatment of bone repair and tissue fibrosis in patients.

Shared decision making in patients at risk of cancer: The role of domain and numeracy

Background
Shared decision making has become an integral part of medical consultation. Research has, however, reported wide differences in individuals' desires to be involved in the decision-making process, and these differences in preferences are likely to be the result of a number of factors including age, education and numeracy.

Objective
To investigate whether patients at genetic risk for cancer had preferences for shared decision making that differed depending on medical domain (general health vs. cancer) and whether decision preferences are linked to numeracy abilities.

Methods
Four hundred and seventy-six women who consented to participate in response to an email sent by a local branch of the U.S.-based Cancer Genetics Network (CGN) to its members. Participants completed the Control Preference Scale, as well as an objective and subjective numeracy scales.

Results
Decision domain (cancer vs. general health) was not associated with women's preferences for involvement in decision making. Objective and subjective numeracy predicted a preference for decision involvement in general, and only objective numeracy was predictive with regard to cancer.

Conclusion
Participants were equally likely to state they wanted to play an active, collaborative or passive role in both medical domains (general and cancer). High-numeracy participants were more likely to express a desire for an active role in general and in case they were diagnosed with cancer.

Practice implications
Health authorities' recommendations to clinicians to include patients in their medical decisions are supported by patients' desires, and clinicians should be cognizant of their patients' preferences as well as their numeracy skills.

Immunoselection of breast and ovarian cancer cells with trastuzumab and natural killer cells:selective escape of CD44high/CD24low/HER2low breast cancer stem cells

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44(high)CD24(low) "cancer stem cell-like" phenotype and expressed significantly less HER2 compared with non-stem cells. Likewise, the CD44(high)CD24(low) population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44(high)CD24(low) cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants.

KIF2A silencing inhibits the proliferation and migration of breast cancer cells and correlates with unfavorable prognosis in breast cancer

Background: Kinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied.

Methods: Immunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients' survival in relation to KIF2A expression was estimated using the Kaplan-Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection.

Results: The expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P <0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P <0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P <0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P <0.05).

Conclusions: KIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer.

Urothelial cancer gene regulatory networks inferred from large-scale RNAseq, Bead and Oligo gene expression data

BACKGROUND: Urothelial pathogenesis is a complex process driven by an underlying network of interconnected genes. The identification of novel genomic target regions and gene targets that drive urothelial carcinogenesis is crucial in order to improve our current limited understanding of urothelial cancer (UC) on the molecular level. The inference of genome-wide gene regulatory networks (GRN) from large-scale gene expression data provides a promising approach for a detailed investigation of the underlying network structure associated to urothelial carcinogenesis.

METHODS: In our study we inferred and compared three GRNs by the application of the BC3Net inference algorithm to large-scale transitional cell carcinoma gene expression data sets from Illumina RNAseq (179 samples), Illumina Bead arrays (165 samples) and Affymetrix Oligo microarrays (188 samples). We investigated the structural and functional properties of GRNs for the identification of molecular targets associated to urothelial cancer.

RESULTS: We found that the urothelial cancer (UC) GRNs show a significant enrichment of subnetworks that are associated with known cancer hallmarks including cell cycle, immune response, signaling, differentiation and translation. Interestingly, the most prominent subnetworks of co-located genes were found on chromosome regions 5q31.3 (RNAseq), 8q24.3 (Oligo) and 1q23.3 (Bead), which all represent known genomic regions frequently deregulated or aberated in urothelial cancer and other cancer types. Furthermore, the identified hub genes of the individual GRNs, e.g., HID1/DMC1 (tumor development), RNF17/TDRD4 (cancer antigen) and CYP4A11 (angiogenesis/ metastasis) are known cancer associated markers. The GRNs were highly dataset specific on the interaction level between individual genes, but showed large similarities on the biological function level represented by subnetworks. Remarkably, the RNAseq UC GRN showed twice the proportion of significant functional subnetworks. Based on our analysis of inferential and experimental networks the Bead UC GRN showed the lowest performance compared to the RNAseq and Oligo UC GRNs.

CONCLUSION: To our knowledge, this is the first study investigating genome-scale UC GRNs. RNAseq based gene expression data is the data platform of choice for a GRN inference. Our study offers new avenues for the identification of novel putative diagnostic targets for subsequent studies in bladder tumors.

Rationale and Means to Target Pro-Inflammatory Interleukin-8 (CXCL8) Signaling in Cancer

It is well established that chronic inflammation underpins the development of a number of human cancers, with pro-inflammatory signaling within the tumor microenvironment contributing to tumor progression and metastasis. CXCL8 is an ELR+ pro-inflammatory CXC-chemokine which mediates its effects via signaling through two G protein-coupled receptors, CXCR1 and CXCR2. Elevated CXCL8-CXCR1/2 signaling within the tumor microenvironment of numerous cancers is known to enhance tumor progression via activation of signaling pathways promoting proliferation, angiogenesis, migration, invasion and cell survival. This review provides an overview of established roles of CXCL8-CXCR1/2 signaling in cancer and subsequently, discusses the possible strategies of targeting CXCL8-CXCR1/2 signaling in cancer, covering indirect strategies (e.g., anti-inflammatories, NFκB inhibitors) and direct CXCL8 or CXCR1/2 inhibition (e.g., neutralizing antibodies, small molecule receptor antagonists, pepducin inhibitors and siRNA strategies). Reports of pre-clinical cancer studies and clinical trials using CXCL8-CXCR1/2-targeting strategies for the treatment of inflammatory diseases will be discussed. The future translational opportunities for use of such agents in oncology will be discussed, with emphasis on exploitation in stratified populations.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer.

MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens.

RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples.

CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Androgen-regulated metabolism and biosynthesis in prostate cancer

Metabolic changes are a well-described hallmark of cancer and are responses to changes in the activity of diverse oncogenes and tumour suppressors. For example, steroid hormone biosynthesis is intimately associated with changes in lipid metabolism and represents a therapeutic intervention point in the treatment of prostate cancer (PCa). Both prostate gland development and tumorigenesis rely on the activity of a steroid hormone receptor family member, the androgen receptor (AR). Recent studies have sought to define the biological effect of the AR on PCa by defining the whole-genome binding sites and gene networks that are regulated by the AR. These studies have provided the first systematic evidence that the AR influences metabolism and biosynthesis at key regulatory steps within pathways that have also been defined as points of influence for other oncogenes, including c-Myc, p53 and hypoxia-inducible factor 1α, in other cancers. The success of interfering with these pathways in a therapeutic setting will, however, hinge on our ability to manage the concomitant stress and survival responses induced by such treatments and to define appropriate therapeutic windows.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Prognostic role of lemur tyrosine kinase-3 germline polymorphisms in adjuvant gastric cancer in Japan and the United States

Lemur tyrosine kinase-3 (LMTK3) was recently identified as an estrogen receptor (ER)-α modulator related to endocrine therapy resistance, and its polymorphisms rs9989661 (T>C) T/T genotype and rs8108419 (G>A) G/G or A/G genotype predicted improved outcomes in breast cancer. Because different predominant ER distributions link to breast and gastric cancer and little is known of the prognostic role of LMTK3 in gastric cancer, this study was carried out to clarify the prognostic role of these polymorphisms in gastric cancer. One-hundred and sixty-nine Japanese and 137 U.S. patients with localized gastric adenocarcinoma were enrolled. Genomic DNA was extracted from blood or tissue, and all samples were analyzed by PCR-based direct DNA sequencing. Overall, these polymorphisms were not associated with survival in both cohorts. When gender was considered, in multivariate analysis, harboring rs9989661 T/T genotype was associated with disease-free survival [HR, 4.37; 95% confidence interval (CI), 2.08-9.18; P < 0.0001] and overall survival (OS; HR, 3.69; 95% CI, 1.65-8.24; P = 0.0014) in the Japanese males and time to recurrence (HR, 7.29; 95% CI, 1.07-49.80; P = 0.043) in the U.S. females. Meanwhile, harboring rs8108419 G/G genotype was associated with OS in the Japanese females (HR, 3.04; 95% CI, 1.08-8.56; P = 0.035) and the U.S. males (HR, 3.39; 95% CI, 1.31-8.80; P = 0.012). The prognostic role of these polymorphisms may be negative in gastric cancer. These findings suggest that the estrogen pathway may play a prognostic role in patients with gastric cancer but this may be dependent on the regional differences both in physiology and genetic alterations of gastric cancer.

Personalised and Precision Medicine in Cancer Clinical Trials: Panacea for Progress or Pandora's Box?

Cancer clinical trials have been one of the key foundations for significant advances in oncology. However, there is a clear recognition within the academic, care delivery and pharmaceutical/biotech communities that our current model of clinical trial discovery and development is no longer fit for purpose. Delivering transformative cancer care should increasingly be our mantra, rather than maintaining the status quo of, at best, the often miniscule incremental benefits that are observed with many current clinical trials. As we enter the era of precision medicine for personalised cancer care (precision and personalised medicine), it is important that we capture and utilise our greater understanding of the biology of disease to drive innovative approaches in clinical trial design and implementation that can lead to a step change in cancer care delivery. A number of advances have been practice changing (e.g. imatinib mesylate in chronic myeloid leukaemia, Herceptin in erb-B2-positive breast cancer), and increasingly we are seeing the promise of a number of newer approaches, particularly in diseases like lung cancer and melanoma. Targeting immune checkpoints has recently yielded some highly promising results. New algorithms that maximise the effectiveness of clinical trials, through for example a multi-stage, multi-arm type design are increasingly gaining traction. However, our enthusiasm for the undoubted advances that have been achieved are being tempered by a realisation that these new approaches may have significant cost implications. This article will address these competing issues, mainly from a European perspective, highlight the problems and challenges to healthcare systems and suggest potential solutions that will ensure that the cost/value rubicon is addressed in a way that allows stakeholders to work together to deliver optimal cost-effective cancer care, the benefits of which can be transferred directly to our patients.

SPLI is a Chemoattractant for Oral and Skin Fibroblasts

Objectives: Unlike adult dermal wounds, the oral mucosa demonstrates preferential healing characterized by rapid remodeling and re-epithelialisation, with minimal scar formation. Secretory leukocyte protease inhibitor (SLPI) is an epithelial-derived factor with potential for promoting scarless repair. The aims of this study were to: (i) investigate the directed migratory (chemotaxis) response of oral and skin fibroblasts to various concentrations of SLPI; and (ii) compare migratory speed of the two cell types. Methods: Paired oral and skin fibroblasts were seeded at 2x104 cells in six well plates containing glass coverslips, and cultured in DMEM supplemented with 10% FCS for 48hours. Following a period of serum starvation (18hours in DMEM plus 0.5% BSA), coverslips were incorporated within a Dunn chemotaxis chamber containing DMEM with 0.5% BSA +/- SLPI gradients at 0.5, 1 or 2µM concentrations. Using microscopy, the migratory behaviour of cells was digitally captured every 10mins for 18hours, traced with JCell tracking software and resulting co-ordinates statistically analysed using Mathmatica software. Results: At all concentrations SLPI was a significant chemoattractant (p<0.01) for both cell types. However, skin fibroblasts migrated significantly faster than oral cells at each SLPI concentration, with greatest effect observed at the highest dose (skin: 32.0±0.47µm/hr, oral: 13.6±0.23µm/hr). Conclusion: SLPI is a chemoattractant for both oral and skin fibroblasts, and may play an important role in fibroblast recruitment during wound healing. This work was funded by the R&D Office, N.Ireland.

Health care professionals’ experience, understanding and perception of need of advanced cancer patients with cachexia and their families: The benefits of a dedicated clinic.

Background: Cachexia has been defined as an on-going loss of skeletal muscle mass that cannot be fully reversed by conventional nutritional support. It can be found in up to 80% of patients with advanced cancer and has profound psycho-social consequences for patients and their families. There is a paucity of studies examining the role and experience of healthcare professionals in relation to cachexia and existing studies suggest that professional staff have limited understanding and do not intervene effectively.
Aim: To identify barriers and facilitators to good practice in cachexia care in order to inform future developments in service provision.
Design: An exploratory qualitative study was conducted employing semi-structured interviews with a range of healthcare professionals recruited purposefully from an Australian hospital. Interviews were conducted in private rooms within the hospital.
Setting/participants: A range of healthcare professionals responsible for cancer care were recruited from a large Australian teaching hospital.
Results: Interviews were conducted with 8 healthcare professionals responsible for delivering cancer care. Four themes were identified: formal and informal education, knowledge and understanding, truth telling in cachexia and palliative care, and, a multi-disciplinary approach. Findings show how improved knowledge and understanding across a staff body can lead to improved staff confidence and a willingness to address cancer cachexia and its consequences with patients and their families.
Conclusion: Comparison with previous studies illustrates the importance of improving knowledge and understanding about cachexia and how this can contribute to staff having the skills and experience necessary to address cachexia and provide an improved care experience for patients and carers.