Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

EBF1, MYO6 and CALR expression levels predict therapeutic response in diffuse large B-cell lymphomas.

Background: Diffuse large B-cell lymphoma (DLBCL) is a hematological malignancy representing one-third of non-Hodgkin's lymphoma cases. Notwithstanding immunotherapy in combination with chemotherapy (R-CHOP) is an effective therapeutic approach for DLBCL, a subset of patients encounters treatment resistance, leading to low survival rates. Thus, there is an urgent need to identify predictive biomarkers for DLBCL including the elderly population, which represents the fastest-growing segment of the population in Western countries. Methods: Gene expression profiles of n=414 DLBCL biopsies were retrieved from the public dataset GSE10846. Differentially expressed genes (DEGs) (fold change >1.4, p-value <0.05, n=387) have been clustered in responder and non-responder patient cohorts. An enrichment analysis has been performed on the top 30 up-regulated genes of responder and non-responder patients to identify the signatures involved in gene ontology (MSigDB). The more significantly up-regulated DEGs have been validated in our independent collection of formalin-fixed paraffin-embedded (FFPE) biopsy samples of elderly DLBCL patients, treated with R-CHOP as first-line therapy. Results: From the analysis of two independent cohorts of DLBCL patients emerged a gene signature able to predict the response to R-CHOP therapy. In detail, expression levels of EBF1, MYO6, CALR are associated with a significant worse overall survival. Conclusions: These results pave the way for a novel characterization of DLBCL biomarkers, aiding the stratification of responder versus non-responder patients.

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation.

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

Destroying the Shield of Cancer Stem Cells: Natural Compounds as Promising Players in Cancer Therapy.

In a scenario where eco-sustainability and a reduction in chemotherapeutic drug waste are certainly a prerogative to safeguard the biosphere, the use of natural products (NPs) represents an alternative therapeutic approach to counteract cancer diseases. The presence of a heterogeneous cancer stem cell (CSC) population within a tumor bulk is related to disease recurrence and therapy resistance. For this reason, CSC targeting presents a promising strategy for hampering cancer recurrence. Increasing evidence shows that NPs can inhibit crucial signaling pathways involved in the maintenance of CSC stemness and sensitize CSCs to standard chemotherapeutic treatments. Moreover, their limited toxicity and low costs for large-scale production could accelerate the use of NPs in clinical settings. In this review, we will summarize the most relevant studies regarding the effects of NPs derived from major natural sources, e.g., food, botanical, and marine species, on CSCs, elucidating their use in pre-clinical and clinical studies.

Mex3a marks drug-tolerant persister colorectal cancer cells that mediate relapse after chemotherapy.

Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.

Targeting of the Peritumoral Adipose Tissue Microenvironment as an Innovative Antitumor Therapeutic Strategy.

The tumor microenvironment (TME) plays a key role in promoting and sustaining cancer growth. Adipose tissue (AT), due to its anatomical distribution, is a prevalent component of TME, and contributes to cancer development and progression. Cancer-associated adipocytes (CAAs), reprogrammed by cancer stem cells (CSCs), drive cancer progression by releasing metabolites and inflammatory adipokines. In this review, we highlight the mechanisms underlying the bidirectional crosstalk among CAAs, CSCs, and stromal cells. Moreover, we focus on the recent advances in the therapeutic targeting of adipocyte-released factors as an innovative strategy to counteract cancer progression.

Dual Inhibition of Myc Transcription and PI3K Activity Effectively Targets Colorectal Cancer Stem Cells.

Despite advances in the curative approach, the survival rate of advanced colorectal cancer (CRC) patients is still poor, which is likely due to the emergence of cancer cell clones resistant to the available therapeutic options. We have already shown that CD44v6-positive CRC stem cells (CR-CSCs) are refractory toward standard anti-tumor therapeutic agents due to the activation of the PI3K pathway together with high HER2 expression levels. Tumor microenvironmental cytokines confer resistance to CR-CSCs against HER2/PI3K targeting by enhancing activation of the MAPK pathway. Here, we show that the CSC compartment, spared by BRAF inhibitor-based targeted therapy, is associated with increased expression levels of CD44v6 and Myc and retains boosted clonogenic activity along with residual tumorigenic potential. Inhibition of Myc transcription, downstream of the MAPK cascade components, and PI3K pathway activity was able to overcome the protective effects of microenvironmental cytokines, affecting the survival and the clonogenic activity of CR-CSCs, regardless of their mutational background. Likewise, the double targeting induced stabilization of mouse tumor avatars. Altogether, these data outline the rationale for dual kinase targeting of CR-CSCs to prevent their adaptive response, which would lead to disease progression.

Dissecting the Mechanism of Action of Spiperone-A Candidate for Drug Repurposing for Colorectal Cancer.

Approximately 50% of colorectal cancer (CRC) patients still die from recurrence and metastatic disease, highlighting the need for novel therapeutic strategies. Drug repurposing is attracting increasing attention because, compared to traditional de novo drug discovery processes, it may reduce drug development periods and costs. Epidemiological and preclinical evidence support the antitumor activity of antipsychotic drugs. Herein, we dissect the mechanism of action of the typical antipsychotic spiperone in CRC. Spiperone can reduce the clonogenic potential of stem-like CRC cells (CRC-SCs) and induce cell cycle arrest and apoptosis, in both differentiated and CRC-SCs, at clinically relevant concentrations whose toxicity is negligible for non-neoplastic cells. Analysis of intracellular Ca2+ kinetics upon spiperone treatment revealed a massive phospholipase C (PLC)-dependent endoplasmic reticulum (ER) Ca2+ release, resulting in ER Ca2+ homeostasis disruption. RNA sequencing revealed unfolded protein response (UPR) activation, ER stress, and induction of apoptosis, along with IRE1-dependent decay of mRNA (RIDD) activation. Lipidomic analysis showed a significant alteration of lipid profile and, in particular, of sphingolipids. Damage to the Golgi apparatus was also observed. Our data suggest that spiperone can represent an effective drug in the treatment of CRC, and that ER stress induction, along with lipid metabolism alteration, represents effective druggable pathways in CRC.

BCL-XL inhibition induces an FGFR4-mediated rescue response in colorectal cancer.

The heterogeneous therapy response observed in colorectal cancer is in part due to cancer stem cells (CSCs) that resist chemotherapeutic insults. The anti-apoptotic protein BCL-XL plays a critical role in protecting CSCs from cell death, where its inhibition with high doses of BH3 mimetics can induce apoptosis. Here, we screen a compound library for synergy with low-dose BCL-XL inhibitor A-1155463 to identify pathways that regulate sensitivity to BCL-XL inhibition and reveal that fibroblast growth factor receptor (FGFR)4 inhibition effectively sensitizes to A-1155463 both in vitro and in vivo. Mechanistically, we identify a rescue response that is activated upon BCL-XL inhibition and leads to rapid FGF2 secretion and subsequent FGFR4-mediated post-translational stabilization of MCL-1. FGFR4 inhibition prevents MCL-1 upregulation and thereby sensitizes CSCs to BCL-XL inhibition. Altogether, our findings suggest a cell transferable induction of a FGF2/FGFR4 rescue response in CRC that is induced upon BCL-XL inhibition and leads to MCL-1 upregulation.

Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51.

Breast cancer (BC) is the second cause of cancer-related deceases in the worldwide female population. Despite the successful treatment advances, 25% of BC develops resistance to current therapeutic regimens, thereby remaining a major hurdle for patient management. Current therapies, targeting the molecular events underpinning the adaptive resistance, still require effort to improve BC treatment. Using BC sphere cells (BCSphCs) as a model, here we showed that BC stem-like cells express high levels of Myc, which requires the presence of the multifunctional DNA/RNA binding protein Sam68 for the DNA-damage repair. Analysis of a cohort of BC patients displayed that Sam68 is an independent negative factor correlated with the progression of the disease. Genetic inhibition of Sam68 caused a defect in PARP-induced PAR chain synthesis upon DNA-damaging insults, resulting in cell death of TNBC cells. In contrast, BC stem-like cells were able to survive due to an upregulation of Rad51. Importantly, the inhibition of Rad51 showed synthetic lethal effect with the silencing of Sam68, hampering the cell viability of patient-derived BCSphCs and stabilizing the growth of tumor xenografts, including those TNBC carrying BRCA mutation. Moreover, the analysis of Myc, Sam68 and Rad51 expression demarcated a signature of a poor outcome in a large cohort of BC patients. Thus, our findings suggest the importance of targeting Sam68-PARP1 axis and Rad51 as potential therapeutic candidates to counteract the expansion of BC cells with an aggressive phenotype.

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Adipose stromal cells promote the transition of colorectal cancer cells toward a mesenchymal-like phenotype.

Colon cancer progression is among the risks that increase with obesity. We have recently unveiled the molecular mechanism by which adipose tissue-released molecules, HGF and IL-6, make colorectal cancer (CRC) cells acquiring mesenchymal traits. Targeting of adipose-derived factors abrogate the metastatic potential of CRC stem cells (CR-CSCs) in obese patients.

Targeting Phosphatases and Kinases: How to Checkmate Cancer.

Metastatic disease represents the major cause of death in oncologic patients worldwide. Accumulating evidence have highlighted the relevance of a small population of cancer cells, named cancer stem cells (CSCs), in the resistance to therapies, as well as cancer recurrence and metastasis. Standard anti-cancer treatments are not always conclusively curative, posing an urgent need to discover new targets for an effective therapy. Kinases and phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality is a hallmark of cancer. Notwithstanding the role of kinases and phosphatases in cancer has been widely investigated, their aberrant activation in the compartment of CSCs is nowadays being explored as new potential Achille's heel to strike. Here, we provide a comprehensive overview of the major protein kinases and phosphatases pathways by which CSCs can evade normal physiological constraints on survival, growth, and invasion. Moreover, we discuss the potential of inhibitors of these proteins in counteracting CSCs expansion during cancer development and progression.

Cancer Stem Cell Biomarkers Predictive of Radiotherapy Response in Rectal Cancer: A Systematic Review.

BACKGROUND: Rectal cancer (RC) is one of the most commonly diagnosed and particularly challenging tumours to treat due to its location in the pelvis and close proximity to critical genitourinary organs. Radiotherapy (RT) is recognised as a key component of therapeutic strategy to treat RC, promoting the downsizing and downstaging of large RCs in neoadjuvant settings, although its therapeutic effect is limited due to radioresistance. Evidence from experimental and clinical studies indicates that the likelihood of achieving local tumour control by RT depends on the complete eradication of cancer stem cells (CSC), a minority subset of tumour cells with stemness properties. METHODS: A systematic literature review was conducted by querying two scientific databases (Pubmed and Scopus). The search was restricted to papers published from 2009 to 2021. RESULTS: After assessing the quality and the risk of bias, a total of 11 studies were selected as they mainly focused on biomarkers predictive of RT-response in CSCs isolated from patients affected by RC. Specifically these studies showed that elevated levels of CD133, CD44, ALDH1, Lgr5 and G9a are associated with RT-resistance and poor prognosis. CONCLUSIONS: This review aimed to provide an overview of the current scenario of in vitro and in vivo studies evaluating the biomarkers predictive of RT-response in CSCs derived from RC patients.

FACS-based protocol to assess cytotoxicity and clonogenic potential of colorectal cancer stem cells using a Wnt/ß-catenin signaling pathway reporter.

Cancer stem cells (CSCs) play a key role in tumor initiation and progression. A real-time tool to evaluate the activation of CSC-specific signaling pathways is crucial for the study of this cancer cell subset. Here, we present a protocol to monitor, in vitro, the activation of Wnt/ß-catenin signaling pathway, which is considered a functional biomarker for colorectal CSCs (CR-CSCs). This flow-cytometry-based protocol allows it to isolate CR-CSCs and to evaluate their cytotoxicity upon anti-tumor treatments. For complete details on the use and execution of this protocol, please refer to Di Franco et al. (2021).

Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery.

Obesity is a strong risk factor for cancer progression, posing obesity-related cancer as one of the leading causes of death. Nevertheless, the molecular mechanisms that endow cancer cells with metastatic properties in patients affected by obesity remain unexplored.Here, we show that IL-6 and HGF, secreted by tumor neighboring visceral adipose stromal cells (V-ASCs), expand the metastatic colorectal (CR) cancer cell compartment (CD44v6 + ), which in turn secretes neurotrophins such as NGF and NT-3, and recruits adipose stem cells within tumor mass. Visceral adipose-derived factors promote vasculogenesis and the onset of metastatic dissemination by activation of STAT3, which inhibits miR-200a and enhances ZEB2 expression, effectively reprogramming CRC cells into a highly metastatic phenotype. Notably, obesity-associated tumor microenvironment provokes a transition in the transcriptomic expression profile of cells derived from the epithelial consensus molecular subtype (CMS2) CRC patients towards a mesenchymal subtype (CMS4). STAT3 pathway inhibition reduces ZEB2 expression and abrogates the metastatic growth sustained by adipose-released proteins. Together, our data suggest that targeting adipose factors in colorectal cancer patients with obesity may represent a therapeutic strategy for preventing metastatic disease.

Nobiletin and Xanthohumol Sensitize Colorectal Cancer Stem Cells to Standard Chemotherapy.

Colorectal cancer (CRC) mortality is mainly caused by patient refractoriness to common anti-cancer therapies and consequent metastasis formation. Besides, the notorious toxic side effects of chemotherapy are a concurrent obstacle to be tackled. Thus, new treatment approaches are needed to effectively improve patient outcomes. Compelling evidence demonstrated that cancer stem cells (CSCs) are responsible for treatment failure and relapse. New natural treatment approaches showed capabilities to selectively target the CSC subpopulation by rendering them targetable by standard cytotoxic compounds. Herein we show the anti-cancer properties of the polymethoxyflavones and prenylflavonoids extracted from Citrus sinensis and Humulus lupulus, respectively. The natural biofunctional fractions, singularly and in combination, reduced the cell viability of CRC stem cells (CR-CSCs) and synergized with 5-fluorouracil and oxaliplatin (FOX) chemotherapy. These phenomena were accompanied by a reduced S and G2/M phase of the cell cycle and upregulation of cell death-related genes. Notably, both phytoextracts in combination with FOX thwarted stemness features in CR-CSCs as demonstrated by the impaired clonogenic potential and decreased Wnt pathway activation. Extracts lowered the expression of CD44v6 and affected the expansion of metastatic CR-CSCs in patients refractory to chemotherapy. Together, this study highlights the importance of polymethoxyflavones and prenylflavonoids as natural remedies to aid oncological therapies.