MicroRNA expression profiling and biomarker validation in treatment-naïve and drug resistant non-small cell lung cancer.

BACKGROUND: In the absence of targetable mutations or immune checkpoints, cisplatin-doublet chemotherapy remains the standard of care in non-small cell lung cancer (NSCLC). Drug resistance has however become a significant clinical challenge. Exploring a role for small non-coding microRNAs (miRNA) as biomarker candidates in cisplatin resistant (CisR) lung cancer is lacking and warrants further investigation. METHODS: miRNA expression profiling was assessed in a panel of cisplatin sensitive and resistant NSCLC cell lines and validated by qPCR. Modulation of altered miRNAs was studied using antagomiRs and pre-miRs while functional assays were used to assess cisplatin response. The translational relevance of these miRNAs as potential biomarkers was assessed in serum and matched normal and tumour lung tissues from chemo-naïve NSCLC patients, in addition to xenograft formalin-fixed paraffin-embedded (FFPE) tumours derived from cisplatin sensitive and resistant cell lines. RESULTS: Differential expression of a 5-miR signature (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286) demonstrated their ability to distinguish between normal and tumour lung tissue and between NSCLC histologies. In squamous cell carcinoma (SqCC), tissue miRNA expression was associated with poor survival. miR-4286 showed promise as a blood-based diagnostic biomarker that could distinguish between adenocarcinoma and SqCC histologies. In a xenograft model of cisplatin resistance, using 7-9 week old female NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl), a 5-miRNA panel showed altered expression between sensitive and resistant tumours. CONCLUSIONS: This study identified a panel of miRNAs which may have diagnostic and prognostic potential as novel biomarkers in lung cancer and furthermore, may have a predictive role in monitoring the emergence of resistance to cisplatin.

Circulating tumour cell enumeration does not correlate with Miller-Payne grade in a cohort of breast cancer patients undergoing neoadjuvant chemotherapy.

PURPOSE: The association between pathological complete response (pCR) in patients receiving neoadjuvant chemotherapy (NAC) for breast cancer and Circulating Tumour Cells (CTCs) is not clear. The aim of this study was to assess whether CTC enumeration could be used to predict pathological response to NAC in breast cancer as measured by the Miller-Payne grading system. METHODS: Twenty-six patients were recruited, and blood samples were taken pre- and post-NAC. CTCs were isolated using the ScreenCell device and stained using a modified Giemsa stain. CTCs were enumerated by 2 pathologists and classified as single CTCs, doublets, clusters/microemboli and correlated with the pathological response as measured by the Miller-Payne grading system. χ2 or ANOVA was performed in SPSS 24.0 statistics software for associations. RESULTS: 89% of patients had invasive ductal carcinoma (IDC) and 11% invasive lobular carcinoma (ILC). At baseline 85% of patients had CTCs present, median 7 (0-161) CTCs per 3 ml of whole blood. Post-chemotherapy, 58% had an increase in CTCs. This did not correlate with the Miller-Payne grade of response. No significant association was identified between the number of CTCs and clinical characteristics; however, we did observe a correlation between pre-treatment CTC counts and body mass index, p < 0.05. CONCLUSIONS: Patients with a complete response to NAC still had CTCs present, suggesting enumeration is not sufficient to aid surgery stratification. Additional characterisation and larger studies are needed to further characterise CTCs isolated pre- and post-chemotherapy. Long-term follow-up of these patients will determine the significance of CTCs in NAC breast cancer patients.

Endogenous exosome labelling with an amphiphilic NIR-fluorescent probe.

The recognition of the biological, diagnostic and medical importance of exosomes has given rise to an urgent need for efficient labelling of these extracellular vesicles in ways that do not alter their inherent characteristics. We report for the first time an endogenous method to NIR-fluorescent labelled exosomes using an amphiphilic probe without the need for immunolabelling or synthetic or chromatographic manipulation of exosomes. Comparative analyses of labelled and unlabelled exosomes with NTA, AFM, flow cytometry and immunoblot analysis all show a high degree of similarity. Spectroscopic analysis and fluorescence imaging confirmed the ability to visualise purified NIR-exosomes.

Targeting the cancer stem cell marker, aldehyde dehydrogenase 1, to circumvent cisplatin resistance in NSCLC.

Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors in vitro. Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of cisplatin.

CD10-/ALDH- cells are the sole cisplatin-resistant component of a novel ovarian cancer stem cell hierarchy.

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10-/ALDH- CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10-/ALDH- CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10-/ALDH- CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10-/ALDH- CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-ß Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Catching moving targets: cancer stem cell hierarchies, therapy-resistance & considerations for clinical intervention.

It is widely believed that targeting the tumour-initiating cancer stem cell (CSC) component of malignancy has great therapeutic potential, particularly in therapy-resistant disease. However, despite concerted efforts, CSC-targeting strategies have not been efficiently translated to the clinic. This is partly due to our incomplete understanding of the mechanisms underlying CSC therapy-resistance. In particular, the relationship between therapy-resistance and the organisation of CSCs as Stem-Progenitor-Differentiated cell hierarchies has not been widely studied. In this review we argue that modern clinical strategies should appreciate that the CSC hierarchy is a dynamic target that contains sensitive and resistant components and expresses a collection of therapy-resisting mechanisms. We propose that the CSC hierarchy at primary presentation changes in response to clinical intervention, resulting in a recurrent malignancy that should be targeted differently. As such, addressing the hierarchical organisation of CSCs into our bench-side theory should expedite translation of CSC-targeting to bed-side practice. In conclusion, we discuss strategies through which we can catch these moving clinical targets to specifically compromise therapy-resistant disease.

MicroRNA-17 is downregulated in esophageal adenocarcinoma cancer stem-like cells and promotes a radioresistant phenotype.

Resistance to neoadjuvant chemoradiation therapy (CRT) remains a critical barrier to the effective treatment of esophageal adenocarcinoma (EAC). Cancer stem-like cells (CSCs) are a distinct subpopulation of cells implicated in the resistance of tumors to anti-cancer therapy. However, their role in the resistance of EAC to CRT is largely unknown. In this study, using a novel in vitro isogenic model of radioresistant EAC, we demonstrate that radioresistant EAC cells have enhanced tumorigenicity in vivo, increased expression of CSC-associated markers and enhanced holoclone forming ability. Further investigation identified a subpopulation of cells that are characterised by high aldehyde dehydrogenase (ALDH) activity, enhanced radioresistance and decreased expression of miR-17-5p. In vitro, miR-17-5p was demonstrated to significantly sensitise radioresistant cells to X-ray radiation and promoted the repression of genes with miR-17-5p binding sites, such as C6orf120. In vivo, miR-17-5p was significantly decreased, whilst C6orf120 was significantly increased, in pre-treatment EAC tumour samples from patients who demonstrated a poor response to neoadjuvant CRT. This study sheds novel insights into the role of CSCs in the resistance of EAC to CRT and highlights miR-17-5p as a potential biomarker of CRT sensitivity and novel therapeutic target in treatment resistant EAC.

Enhanced regulation of cell cycle and suppression of osteoblast differentiation molecular signatures by prostate cancer stem-like holoclones.

AIMS: Targeting the stem cell properties of tumor-initiating cells is an avenue through which cancer treatment may be improved. Before this can be achieved, so-called 'cancer stem cell' (CSC) models must be developed and characterized in specific malignancies. METHODS: In this study, holoclone formation assays were used to characterise stem-like molecular signatures in prostate cancer (PCa) cells. RESULTS: LNCaP and PC3 parent cells were capable of responding to stem cell differentiation morphogen retinoic acid (RA), suggesting the presence of inherent stem-like properties. LNCaP cells, which represent early, androgen-responsive disease, formed holoclones after twenty six days. PC3 cells, which represent advanced, metastatic, castration-resistant disease, formed holoclones after only six days. Holoclones displayed decreased expression of RA-genes, suggesting a more immature, less differentiated phenotype. Gene and microRNA arrays demonstrated that holoclones downregulated a number of stem cell differentiation regulators while displaying enhanced regulation of G2 to M transition and the mitotic spindle checkpoint components of the cell cycle. PC3 holoclones displayed pronounced downregulation of known regulators of osteoblast differentiation from mesenchymal stem cells and Epithelial Mesenchymal Transition. CONCLUSIONS: Our results suggest that some PCa cells retain the ability to transition to a more immature state in which differentiation and metastatic mechanisms are suppressed. The highlighting of osteoblast differentiation regulators in this mechanism is particularly notable, considering the propensity of PCa to metastasise to bone.

Developing ovarian cancer stem cell models: laying the pipeline from discovery to clinical intervention.

Despite decades of research, ovarian cancer is still associated with unacceptably high mortality rates, which must be addressed by novel therapeutic approaches. One avenue through which this may be achieved is targeting of tumor-initiating 'Cancer Stem Cells' (CSCs). CSCs are sufficient to generate primary and recurrent disease through extensive rounds of asymmetric division, which maintain the CSC pool while producing the tissues that form the bulk of the tumor. CSCs thrive in the harsh tumor niche, are generally refractory to therapeutic intervention and closely-linked to the Epithelial-Mesenchymal Transition process, which facilitates invasion and metastasis. While it is well-accepted that CSC-targeting must be assessed as a novel therapeutic avenue, few ovarian CSC models have been developed due to perceived and actual difficulties associated with the process of 'CSC Discovery'. In this article we review contemporary approaches to CSC Discovery and argue that this process should start with an understanding of the specific challenges associated with clinical intervention, laying the pipeline backwards towards CSC Discovery. Such an approach would expedite the bridging of the gap between laboratory isolation and clinical targeting of ovarian CSCs.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer.

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Suppression of cancer stemness p21-regulating mRNA and microRNA signatures in recurrent ovarian cancer patient samples.

BACKGROUND: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. METHODS: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. RESULTS: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. CONCLUSION: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells.

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.