Cellular localization of nucleolin determines the prognosis in cancers: a meta-analysis

Nucleolin (NCL) is a multifunctional protein expressed in the nucleus, cytoplasm, and cell membrane. Overexpression of NCL has a controversial role as a poor prognostic marker in cancers. In this study, a meta-analysis was performed to evaluate the prognostic value of NCL in different subcellular localizations (cytoplasmic (CyNCL) and nuclear (NuNCL)) across a range of cancers. PubMed was searched for relevant publications. Data were extracted and analyzed from 12 studies involving 1221 patients with eight cancer types. The results revealed high total NCL was significantly associated with poor overall survival (OS) (HR = 2.85 (1.94, 4.91), p < 0.00001, I2 = 59%) and short disease-free survival (DFS) (HR = 3.57 (2.76, 4.62), p < 0.00001, I2 = 2%). High CyNCL was significantly associated with poor OS (HR = 4.32 (3.01, 6.19), p < 0.00001, I2 = 0%) and short DFS (HR = 3.00 (2.17, 4.15), p < 0.00001, I2 = 0%). In contrast, high NuNCL correlated with increased patient OS (HR = 0.42 (0.20, 0.86), p = 0.02, I2 = 66%), with no significant correlation to DFS observed (HR = 0.46 (0.19, 1.14), p = 0.09, I2 = 57%). This study supports the role of subcellular NCL as a poor prognostic cancer biomarker

High expression of STAT3 within the tumour-associated stroma predicts poor outcome in breast cancer patients

Introduction: Triple-negative breast cancer (TNBC) patients have the poorest clinical outcomes compared to other molecular subtypes of breast cancer. IL6/JAK/STAT3 signalling is upregulated in breast cancer; however, there is limited evidence for its role in TNBC. This study aimed to assess the expression of IL6/JAK/STAT3 in TNBC as a prognostic biomarker. Methods: Tissue microarrays consisting of breast cancer specimens from a retrospective cohort (n = 850) were stained for IL6R, JAK1, JAK2 and STAT3 via immunohistochemistry. Staining intensity was assessed by weighted histoscore and analysed for association with survival/clinical characteristics. In a subset of patients (n = 14) bulk transcriptional profiling was performed using TempO-Seq. Nanostring GeoMx® digital spatial profiling was utilised to establish the differential spatial gene expression in high STAT3 tumours. Results: In TNBC patients, high expression of stromal STAT3 was associated with reduced cancer-specific survival (HR = 2.202, 95% CI: 1.148–4.224, log rank p = 0.018). TNBC patients with high stromal STAT3 had reduced CD4+ T-cell infiltrates within the tumour (p = 0.001) and higher tumour budding (p = 0.003). Gene set enrichment analysis (GSEA) of bulk RNA sequencing showed high stromal STAT3 tumours were characterised by enrichment of IFNγ, upregulation of KRAS signalling and inflammatory signalling Hallmark pathways. GeoMx™ spatial profiling showed high stromal STAT3 samples. Pan cytokeratin (panCK)-negative regions were enriched for CD27 (p < 0.001), CD3 (p < 0.05) and CD8 (p < 0.001). In panCK-positive regions, high stromal STAT3 regions had higher expression of VEGFA (p < 0.05). Conclusion: High expression of IL6/JAK/STAT3 proteins was associated with poor prognosis and characterised by distinct underlying biology in TNBC

Spatial transcriptomic analysis of tumour with high and low CAIX expression in TNBC tissue samples using GeoMx™ RNA assay

Purpose. Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients. Methods. Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients.In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P<0.05 and fold change ≥1 or ≤-1 were identified. Four genes were validated at the protein level. Result. Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis. Conclusions. DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted

Histopathological tumour microenvironment score independently predicts outcome in primary operable colorectal cancer

Colorectal cancer (CRC) is a heterogenous malignancy and research is focused on identifying novel ways to subtype patients. In this study, a novel classification system, tumour microenvironment score (TMS), was devised based on Klintrup–Mäkinen grade (KMG), tumour stroma percentage (TSP), and tumour budding. TMS was performed using a haematoxylin and eosin (H&E)-stained section from retrospective CRC discovery and validation cohorts (n = 1,030, n = 787). TMS0 patients had high KMG, TMS1 were low for KMG, TSP, and budding, TMS2 were high for budding, or TSP and TMS3 were high for TSP and budding. Scores were assessed for association with survival and clinicopathological characteristics. Mutational landscaping and Templated Oligo-Sequencing (TempO-Seq) profiling were performed to establish differences in the underlying biology of TMS. TMS was independently prognostic in both cohorts (p < 0.001, p < 0.001), with TMS3 predictive of the shortest survival times. TMS3 was associated with adverse clinical features including sidedness, local and distant recurrence, higher T stage, higher N stage, and presence of margin involvement. Gene set enrichment analysis of TempO-Seq data showed higher expression of genes associated with hallmarks of cancer pathways including epithelial to mesenchymal transition (p < 0.001), IL2 STAT5 signalling (p = 0.007), and angiogenesis (p = 0.017) in TMS3. Additionally, enrichment of immunosuppressive immune signatures was associated with TMS3 classification. In conclusion, TMS represents a novel and clinically relevant method for subtyping CRC patients from a single H&E-stained tumour section

TAK1 expression is associated with increased PD-L1 and decreased cancer-specific survival in microsatellite-stable colorectal cancer

Background: Transforming growth factor β-activated protein kinase-1 (TAK1) plays an important role in MAPK and NFκB pathways and has been associated with colorectal cancer. The aim of this study was to determine how cytoplasmic and juxtanuclear punctate staining of TAK1 relates to immune checkpoint expression and cancer specific survival in colorectal cancer. Methods: Protein expression was assessed by immunohistochemistry on tissue microarrays from primary curative colorectal cancer resected specimens. Expression levels of cytoplasmic TAK1 by QuPath digital quantification and punctate TAK1 staining was scored using a manual point scoring technique and correlated with clinicopathological features, immune checkpoint expression and cancer-specific survival. Bulk RNA sequencing was performed in specimens to determine mutational profiles and differentially expressed genes. Results: A cohort of 875 patients who had undergone colorectal cancer resection were assessed for TAK1 expression. Higher levels of cytoplasmic TAK1 expression correlated with elevated PD1 and PD-L1 expression (p < 0.010). High punctate TAK1 expression was more commonly identified in poorly differentiated colorectal cancers (p = 0.036), had dysregulated mutational and transcriptional profiles with decreased insulin-like growth factor 2(IGF2) expression (p < 0.010), and independently predicted poor cancer-specific survival (HR 2.690, 95% CI 1.419–5.100, p = 0.002). The association of punctate TAK1 expression and recurrence remained after subgroup analysis for microsatellite-stable colorectal cancer (p = 0.028). Discussion: Punctate TAK1 expression is associated with worse cancer specific survival. TAK1 signalling may be an important pathway to investigate underlying mechanisms for recurrence in microsatellite-stable colorectal cancer

JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC

The molecular mechanism of tumour budding and its relationship with tumour microenvironment in colorectal cancer

Summary Colorectal cancer (CRC) is the third most diagnosed cancer and the second lethal disease worldwide (1). CRC development has recently been well-documented, and the screening program has been shown to improve patient outcomes and survival due to the early detection of the disease (2, 3). However, some patients still experienced disease metastasis with a 5 year survival of only 12% (4). Recent studies have focused on identifying prognostic biomarkers that can predict the adverse outcomes in CRC patients (5). One promising factor that has recently been reported is tumour budding (TB). TB, the single or up to four tumour cells found at the invasive tumour area, is now a well-known prognostic independent biomarker in many solid cancers including CRC (6). Patients with high TB phenotype experienced a poor outcome with an incidence of disease recurrence and metastasis (7). In 2016, the international tumour budding consensus conference (ITBCC) was held and agreed to set up the criteria for TB assessment and suggested to include TB status in a routine clinical report (8). Since then, multiple studies have investigated the prognostic role of TB not only in CRC but also in other solid cancers such as pancreatic (9), breast (10, 11), head and neck (12) and lung (13) cancer. Although TB has a strong prognostic value, few studies investigated its underlying mechanism and how it may relate to adverse features and disease metastasis in CRC. It has been hypothesised that TB could undergo epithelial-mesenchymal transition (EMT), thereby, allowing cells to escape from the main tumour and promote metastasis (14, 15). However, some studies argued that TB may only undergo partial EMT and there is another tumour-related signalling involved in its formation and induction of the metastasis (16, 17). Moreover, some studies have reported an inverse correlation between TB and cytotoxic T cells which could suggested an immunosuppressive role of TB leading to disease metastasis in CRC (18-20). Until now, there has been little understanding of the underlying mechanism of TB and its relationship with the tumour microenvironment in CRC (21). This thesis aims to unravel the molecular mechanism of TB to identify the potential tumour signalling that drives TB formation and how TB is associated with the immune profile at the invasive edge of the tumour. To investigate this, TB status in CRC patients has been identified according to the TB assessment criteria from ITBCC. After that, bulk transcriptomic RNA (n=787) was used to identify tumour-related signalling expressed in tumours with high TB phenotype compared to low TB group. In addition, regional bulk spatial transcriptomic (GeoMx) (n=12) was performed to identify gene expression within the region of interested (ROI), the classification of tumour and stromal areas using specific protein mask (PanCK+/-) was done. This allows the identification of the potential genes related to both budding tumour cells and the surrounded tumour microenvironment between tumours with low and high TB profile as well as the different area of interest (AOI); tumour core, invasive edge, distant stromal area, within the same tumours. The results from GeoMx were later validated in a TMA of the full CRC cohort (n=787), using immunohistochemistry, to verify the translation from RNA to protein. Of these, cyclinD1 expression within TB was identified as a promising prognostic value in CRC patients. Additionally, multiplex immunofluorescence (mIF) using immune panels (lymphocytes and myeloid cells) were also performed to investigate the immune profile within the invasive budding area. Results showed a high density of regulatory and low cytotoxic T cells within the invasive compared to further stromal area of tumours with high TB. Nearest neighbour analysis also showed that TB tend to have a closer distant to regulatory cells as well as pan-macrophages. This finding suggested that TB may have a possible interaction with the surrounding immune cells leading to an alteration of the microenvironment to help it thrive and invade other parts of the body. To investigate if TB formation can be observed within an in vitro setting, CRC spheroids were cultured. The induction of TNF-α and TGF-β were shown to stimulate more TB formation in CRC spheroids and that cyclinD1 expression within the TB was higher in treated spheroids compared to control groups. Moreover, mouse AKPT organoids showed an increased in roundness, which indicates disruption in the formation of TB, in treated compared to control groups. These results suggested that cyclinD1 expressed within TB could have a potential role as a prognostic marker and may be used as a biomarker for TB formation. In summary, data from this thesis have demonstrated potential biomarkers of TB and the relationship with tumour microenvironment in CRC. This will help understand the underlying mechanism of TB, and how they might interact with the surrounding microenvironment and could also pave the way for a future target therapeutic approach in CR

Spatial transcriptomic analysis of tumoural (PanCK+) and stromal (PanCK-) regions in TNBC tissue samples using an immunoncology probe-based panel on the GeoMx™ Digital Spatial Profiler (DSP)

This dataset was obtained by in-situ hybridisation of probes to a tissue microarray (TMA) of human triple-negative breast cancer (TNBC) tissue. The purpose of this dataset is to allow interrogation of the differential transcriptomic profiles of tumoural (PanCK+) and stromal (PanCK-) regions of TNBC disease to further investigate disease pathogenesis and spark hypothesis-driven research into potential new therapies and prognostic markers for patients with this subtype of disease

Systematic review of tumour budding and association with common mutations in patients with colorectal cancer

Introduction: Despite a well-known prognostic role in colorectal cancer, the genomic profiling of tumour budding remains to be elucidated. We aim to review the association of common mutations with tumour budding. Methods: A systematic review of studies relating to tumour budding and genetic mutation in CRC was performed. The relationship between mutational status and tumour budding was evaluated using meta-analysis. Results: A total of 6153 patients from 17 articles were included. According to the meta-analysis, high-grade tumour budding was significantly associated with KRAS mutation (OR = 1.52, 95%CI: 1.13-2.02, P = 0.005) and MSS/pMMR (OR = 2.06, 95%CI: 1.42-2.97, P = 0.0001). Conclusion: The significant association between high-grade tumour budding and mutated KRAS or MSS/pMMR may suggest a role of these mutations in the development of the tumour budding phenotype and be useful for stratifying patient outcome in CRC