Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/ß-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

A Necessary Role for Increased Biglycan Expression during L1-Mediated Colon Cancer Progression.

Aberrant activation of Wnt/ß-catenin signaling and downstream ß-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell-cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors.

Recent insights into the role of L1CAM in cancer initiation and progression.

First described as a neuronal cell adhesion molecule, L1CAM was later identified to be present at increased levels in primary tumors and metastases of various types of cancer. Here, we describe the multifaceted roles of L1CAM that are involved in diverse fundamental steps during tumor initiation and progression, as well as in chemoresistance. Recently, Ganesh et al reported that L1CAM identifies metastasis-initiating cells in colorectal carcinoma exhibiting stem-like cell features, increased tumorigenic potential and enhanced chemoresistance. In this review, we highlight recent advances in L1CAM research with particular emphasis on its role in de-differentiation processes and cancer cell stemness supporting the view that L1CAM is a powerful prognostic factor and a suitable target for improved therapy of metastatic and drug-resistant tumors.

The lipid-transfer protein Nir2 enhances epithelial-mesenchymal transition and facilitates breast cancer metastasis.

The involvement of epithelial-mesenchymal transition (EMT) in breast cancer metastasis has been demonstrated in many studies. However, the intracellular proteins and signaling pathways that regulate EMT have not been fully identified. Here, we show that the lipid-transfer protein Nir2 (also known as PITPNM1) enhances EMT in mammary epithelial and breast cancer cells. Nir2 overexpression decreases the expression of epithelial markers and concomitantly increases the expression of mesenchymal markers, whereas silencing of Nir2 expression by small hairpin RNA (shRNA) has opposite effects. Additionally, Nir2 expression is increased during EMT and affects cell morphology, whereas Nir2 depletion attenuates growth factor-induced cell migration. These effects of Nir2 on EMT-associated processes are mainly mediated through the PI3K/AKT and the ERK1/2 pathways. Nir2 depletion also inhibits cell invasion in vitro and lung metastasis in animal models. Immunohistochemical analysis of breast cancer tissue samples reveals a correlation between high Nir2 expression and tumor grade, and Kaplan-Meier survival curves correlate Nir2 expression with poor disease outcome. These results suggest that Nir2 not only enhances EMT in vitro and breast cancer metastasis in animal models, but also contributes to breast cancer progression in human patients.

The phosphatidylinositol-transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling.

Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)-stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)-transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C-terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF-stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA-binding capability and PI-transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.

A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.

Introduction: Ex vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy. Methods: A multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 µM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients' clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST). Results: The cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97) and a sensitivity of 96% (24/25, 95% CI 0.80-0.99). Conclusion: EVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.

Spectrum of Response to Platinum and PARP Inhibitors in Germline BRCA-Associated Pancreatic Cancer in the Clinical and Preclinical Setting.

Germline BRCA-associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti-PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. SIGNIFICANCE: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development. This article is highlighted in the In This Issue feature, p. 1749.

Nuclear factor-kappaB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells.

Hyperactivation of beta-catenin-T-cell-factor (TCF)-regulated gene transcription is a hallmark of colorectal cancer (CRC). The cell-neural adhesion molecule L1CAM (hereafter referred to as L1) is a target of beta-catenin-TCF, exclusively expressed at the CRC invasive front in humans. L1 overexpression in CRC cells increases cell growth and motility, and promotes liver metastasis. Genes induced by L1 are also expressed in human CRC tissue but the mechanisms by which L1 confers metastasis are still unknown. We found that signaling by the nuclear factor kappaB (NF-kappaB) is essential, because inhibition of signaling by the inhibitor of kappaB super repressor (IkappaB-SR) blocked L1-mediated metastasis. Overexpression of the NF-kappaB p65 subunit was sufficient to increase CRC cell proliferation, motility and metastasis. Binding of the L1 cytodomain to ezrin - a cytoskeleton-crosslinking protein - is necessary for metastasis because when binding to L1 was interrupted or ezrin gene expression was suppressed with specific shRNA, metastasis did not occur. L1 and ezrin bound to and mediated the phosphorylation of IkappaB. We also observed a complex containing IkappaB, L1 and ezrin in the juxtamembrane region of CRC cells. Furthermore, we found that L1, ezrin and phosphorylated p65 are co-expressed at the invasive front in human CRC tissue, indicating that L1-mediated activation of NF-kappaB signaling involving ezrin is a major route of CRC progression.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1.

The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1-ezrin-NF-κB signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-κB signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

Ex vivo organotypic cultures for synergistic therapy prioritization identify patient-specific responses to combined MEK and Src inhibition in colorectal cancer.

Translating preclinical studies to effective treatment protocols and identifying specific therapeutic responses in individuals with cancer is challenging. This may arise due to the complex genetic makeup of tumor cells and the impact of their multifaceted tumor microenvironment on drug response. To find new clinically relevant drug combinations for colorectal cancer (CRC), we prioritized the top five synergistic combinations from a large in vitro screen for ex vivo testing on 29 freshly resected human CRC tumors and found that only the combination of mitogen-activated protein kinase kinase (MEK) and proto-oncogene tyrosine-protein kinase Src (Src) inhibition was effective when tested ex vivo. Pretreatment phosphorylated Src (pSrc) was identified as a predictive biomarker for MEK and Src inhibition only in the absence of KRASG12 mutations. Overall, we demonstrate the potential of using ex vivo platforms to identify drug combinations and discover MEK and Src dual inhibition as an effective drug combination in a predefined subset of individuals with CRC.

IRS1 phosphorylation underlies the non-stochastic probability of cancer cells to persist during EGFR inhibition therapy.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations.

L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers.

Aberrant beta-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of beta-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.

Predicting and affecting response to cancer therapy based on pathway-level biomarkers.

Identifying robust, patient-specific, and predictive biomarkers presents a major obstacle in precision oncology. To optimize patient-specific therapeutic strategies, here we couple pathway knowledge with large-scale drug sensitivity, RNAi, and CRISPR-Cas9 screening data from 460 cell lines. Pathway activity levels are found to be strong predictive biomarkers for the essentiality of 15 proteins, including the essentiality of MAD2L1 in breast cancer patients with high BRCA-pathway activity. We also find strong predictive biomarkers for the sensitivity to 31 compounds, including BCL2 and microtubule inhibitors (MTIs). Lastly, we show that Bcl-xL inhibition can modulate the activity of a predictive biomarker pathway and re-sensitize lung cancer cells and tumors to MTI therapy. Overall, our results support the use of pathways in helping to achieve the goal of precision medicine by uncovering dozens of predictive biomarkers.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

The cell adhesion nectin-like molecules (Necl) 1 and 4 suppress the growth and tumorigenic ability of colon cancer cells.

A key step in human colon cancer development includes the hyperactivation of Wnt/beta-catenin signaling and the induction of beta-catenin-TCF target genes that participate in colon cancer progression. Recent studies identified members of the immunoglobulin-like cell adhesion molecules (IgCAM) of the L1CAM family (L1 and Nr-CAM) as targets of beta-catenin-TCF signaling in colon cancer cells. L1 was detected at the invasive front of colon cancer tissue and confers metastasis when overexpressed in cells. In contrast to L1, we did not detect in colon cancer cells significant levels of another IgCAM family of molecules, the nectin-like (Necl) receptors Necl1 and Necl4, while Necl4 was previously found in the normal small intestine and colon tissues. We studied the properties of colon cancer cells in which Necl4 and Necl1 were expressed either alone, or in combination, and found that such cells display a wide range of properties associated with tumor suppression. Expression of both Necl1 and Necl4 was the most efficient in suppressing the tumorigenicity of colon cancer cells. This was associated with enhanced rates of apoptosis and change in several apoptosis-related markers. In contrast to its capacity to suppress tumorigenesis, Necl4 was unable to affect the highly malignant and metastatic capacities of colon cancer cells in which L1 was overexpressed. Our results suggest that various IgCAM receptor families play different roles in affecting the tumorigenic function of the same cells, and that Necl1 and Necl4 can fulfill a tumor suppressive role.

Epithelial-mesenchymal transition and the invasive potential of tumors.

The development of metastasis requires the movement and invasion of cancer cells from the primary tumor into the surrounding tissue. To acquire such invasive abilities, epithelial cancer cells must undergo several phenotypic changes. Some of these, including alterations in cell adhesion and migration, are reminiscent of those observed during the developmental process termed epithelial-mesenchymal transition (EMT). Several master gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. In particular, the regulation of cell adhesion molecules and the signaling pathways linking them to mechanisms of gene regulation has emerged as an important determinant of tumor cell invasion and metastasis. A deeper understanding of these mechanisms should allow both better diagnosis and the development of specific treatments for invasive cancer.

Fascin, a novel target of beta-catenin-TCF signaling, is expressed at the invasive front of human colon cancer.

Cancer cells become metastatic by acquiring a motile and invasive phenotype. This step requires remodeling of the actin cytoskeleton and the expression of exploratory, sensory organelles known as filopodia. Aberrant beta-catenin-TCF target gene activation plays a major role in colorectal cancer development. We identified fascin1, a key component of filopodia, as a target of beta-catenin-TCF signaling in colorectal cancer cells. Fascin1 mRNA and protein expression were increased in primary cancers in a stage-dependent manner. Fascin1 was exclusively localized at the invasive front of tumors also displaying nuclear beta-catenin. Forced expression of fascin1 in colorectal cancer cells increased their migration and invasion in cell cultures and caused cell dissemination and metastasis in vivo, whereas suppression of fascin1 expression by small interfering RNA reduces cell invasion. Although expression of fascin1 in primary tumors correlated with the presence of metastases, fascin1 was not expressed in metastases. Our studies show that fascin1 expression is tightly regulated during development of colon cancer metastases and is a novel target of beta-catenin-TCF signaling. We propose that transient up-regulation of fascin1 in colorectal cancer promotes the acquisition of migratory and invasive phenotypes that lead to metastasis. Moreover, the expression of fascin1 is down-regulated when tumor cells reach their metastatic destination where migration ceases and proliferation is enhanced. Although metastasis to vital organs is often the cause of mortality, only limited success has been attained in developing effective therapeutics against metastatic disease. We propose that genes involved in cell migration and invasion, such as fascin1, could serve as novel targets for metastasis prevention.