MicroRNA-330-5p as a putative modulator of neoadjuvant chemoradiotherapy sensitivity in oesophageal adenocarcinoma

Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of miR-330-5p as a predictive biomarker of patient sensitivity to neo-CRT and as a novel therapeutic target for manipulating cellular sensitivity to neo-CRT in patients with OAC

Investigating the role of microRNAs as modulators of sensitivity to neoadjuvant chemoradiation therapy in oesophageal adenocarcinoma patients

Oesophageal cancer is the eight most common cancer and the sixth leading cause of deaths worldwide. There are two major histological subtypes of oesophageal cancer, with the most predominant subtype in Europe and the USA being oesophageal adenocarcinoma (OAC). The standard of care for OAC patients with locally advanced disease is neoadjuvant therapy and surgical resection. Unfortunately, ~70% of patients do not respond to neoadjuvant therapy and non-responders gain no benefit from the aggressive treatment regimen whilst compromising their quality of life.There is an unmet clinical need for biomarkers predicative of patient’s response to neoadjuvant chemoradiation therapy (neo-CRT). In a pre-treatment setting, predictive biomarkers indicative of patient response could enable the stratification of patients and would ensure individual patients receive the most effective treatment. However, the greater clinical benefit for patients may come from the development of new or combination therapeutics for OAC. Novel chemosensitising and radiosensitising therapeutics could be administered with neo-CRT to enhance tumour sensitivity, improve CRT efficacy and increase survival rates for OAC patients.MicroRNAs (miRNA/miRs) are short non-coding RNA that function to regulate gene expression at the post-transcriptional level. A single miRNA can potentially target hundreds or thousands of mRNA and subsequently alter the expression of multiple genes and proteins. As essential regulators of gene expression, miRNA are involved in all cellular processes and are dysregulated in cancer and other diseases. Furthermore, miRNA have been identified as predictors and modifiers of tumour sensitivity to chemotherapy and radiotherapy in numerous cancer types. Playing a causal role in disease development and progression, miRNA are promising biomarkers and therapeutic targets.In this study miRNA were investigated as biomarkers of OAC patient response to neo-CRT and as potential therapeutic targets through which to enhance tumour sensitivity to neo-CRT. In pre-treatment OAC biopsies, 67 miRNA that were differentially expressed between responders and non-responders to neo-CRT were identified. MiR-330-5p and miR-187 were downregulated in the pre-treatment biopsy samples of the neo-CRT non-responders. The functional roles of miR-330-5p and miR-187 were investigated as modulators of tumour sensitivity to CRT. In vitro the silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to radiation. Furthermore, silencing miR-330-5p altered the expression of extracellular proteases and protease inhibitors, including MMP1. In vivo a pilot study indicated miR-330-5p silencing enhanced tumour growth and may alter tumour sensitivity to cisplatin. In vitro miR-187 overexpression enhanced cellular sensitivity to radiotherapy and cisplatin, implying that the downregulated miR-187 expression in the non-responders may confer resistance to CRT. Furthermore, miR-187 induced apoptosis in vitro and induction of apoptosis is a potential mechanism by which miR-187 enhances radiosensitivity. MiR-187 altered the expression of genes encoding extracellular proteins, including C3 and other immune related genes. Both miR-330-5p and miR-187 are potential regulators of the secretome, thus emphasising the role of miRNA as modulators of the tumour microenvironment. This study has identified miR-330-5p and miR-187 as potential therapeutic targets that could augment OAC tumour sensitivity to neo-CRT

Repurposing FDA approved drugs as radiosensitizers for treating hypoxic prostate cancer

Abstract Background The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. Methods Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. Results Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. Conclusion Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer

Development and Validation of a 28-gene Hypoxia-related Prognostic Signature for Localized Prostate Cancer.

BACKGROUND: Hypoxia is associated with a poor prognosis in prostate cancer. This work aimed to derive and validate a hypoxia-related mRNA signature for localized prostate cancer. METHOD: Hypoxia genes were identified in vitro via RNA-sequencing and combined with in vivo gene co-expression analysis to generate a signature. The signature was independently validated in eleven prostate cancer cohorts and a bladder cancer phase III randomized trial of radiotherapy alone or with carbogen and nicotinamide (CON). RESULTS: A 28-gene signature was derived. Patients with high signature scores had poorer biochemical recurrence free survivals in six of eight independent cohorts of prostatectomy-treated patients (Log rank test P \u3c .05), with borderline significances achieved in the other two (P \u3c .1). The signature also predicted biochemical recurrence in patients receiving post-prostatectomy radiotherapy (n = 130, P = .007) or definitive radiotherapy alone (n = 248, P = .035). Lastly, the signature predicted metastasis events in a pooled cohort (n = 631, P = .002). Prognostic significance remained after adjusting for clinic-pathological factors and commercially available prognostic signatures. The signature predicted benefit from hypoxia-modifying therapy in bladder cancer patients (intervention-by-signature interaction test P = .0026), where carbogen and nicotinamide was associated with improved survival only in hypoxic tumours. CONCLUSION: A 28-gene hypoxia signature has strong and independent prognostic value for prostate cancer patients

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

From Springer Nature via Jisc Publications RouterHistory: received 2020-06-16, accepted 2020-09-28, registration 2020-10-01, online 2020-10-14, pub-electronic 2020-10-14, collection 2020-12Publication status: PublishedFunder: NIHRFunder: Mission Sector of the Egyptian Ministry of Higher Education and Scientific Research.Funder: CRUK; Grant(s): C147/A25254Abstract: Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR

Independence of HIF1a and androgen signaling pathways in prostate cancer

Funder: Cancer Research UK; doi: http://dx.doi.org/10.13039/501100000289Abstract: Background: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. Methods: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. Results: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. Conclusions: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted

Alterations in chemo- and radiotherapy sensitivity with miR-330-5p silencing.

<p>(<b>A</b>) Silencing miR-330-5p (miRZIP-330-5p) in the OE33 cell line did not significantly alter cellular sensitivity to cisplatin or 5-FU compared to the control (miRZIP-VC). However, there was a significant increase in resistance to radiotherapy with miR-330 silencing. Analysis was performed using paired t-test; *<i>p</i> < 0.05. (<b>B</b>) Silencing miR-330-5p in the OE19 cell line did not significantly alter cellular sensitivity to cisplatin, 5-FU or radiation. Analysis was performed using paired t-test. Data are presented as the mean ± SEM.</p

miR-330-5p expression in pre-treatment diagnostic OAC tumour biopsies from responders vs. non-responders to neo-CRT.

<p>(<b>A</b>) MiR-330-5p expression is significantly lower in patients who do not respond to neo-CRT (TRG 4 and 5) when compared with responders (TRG 1 and 2). The two outlier values in the responder data set came from the two patients who were not clinical stage TNM T3. These two outlier values were biopsy specimens derived from tumours graded T<i>is</i> and T2. Analysis was performed using the Mann Whitney U-test; **<i>p</i><0.01. (<b>B</b>) MiR-330-5p expression is significantly lower in patients with TRG 4 (non-responders) compared to patients with TRG 1 and 2 (responders). Analysis was performed using the Mann Whitney U-test; *<i>p</i> < 0.05. Data are presented as the mean ± SEM.</p

miR-330 overexpression does not alter chemosensitivity.

<p>The clonogenic survival assay was used to assess alterations in cellular sensitivity to cisplatin and 5-FU with miR-330 overexpression. The approximate IC₅₀ doses of cisplatin and 5-FU were used to treat the cells for 24 h. The overexpression of miR-330 did not significantly alter cellular sensitivity to cisplatin or 5-FU. Analysis was performed using paired t-test. Data are presented as the mean ± SEM.</p