Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6.

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature.

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

PGC1α confers specificity-metabolic stress and p53-dependent transcription.

In this issue of Molecular Cell, Sen et al. (2011) report the involvement of PGC1α in modulating the transcriptional activity of p53 in metabolically challenged cells. They provide important insights into the mechanisms linking length and strength of the metabolic stress stimuli to the specific activation of p53 target genes.

MicroRNAs: Non-coding fine tuners of receptor tyrosine kinase signalling in cancer.

Emerging evidence point to a crucial role for non-coding RNAs in modulating homeostatic signaling under physiological and pathological conditions. MicroRNAs, the best-characterized non-coding RNAs to date, can exquisitely integrate spatial and temporal signals in complex networks, thereby confer specificity and sensitivity to tissue response to changes in the microenvironment. MicroRNAs appear as preferential partners for Receptor Tyrosine Kinases (RTKs) in mediating signaling under stress conditions. Stress signaling can be especially relevant to disease. Here we focus on the ability of microRNAs to mediate RTK signaling in cancer, by acting as both tumor suppressors and oncogenes. We will provide a few general examples of microRNAs modulating specific tumorigenic functions downstream of RTK signaling and integrate oncogenic signals from multiple RTKs. A special focus will be devoted to epidermal growth factor receptor (EGFR) signaling, a system offering relatively rich information. We will explore the role of selected microRNAs as bidirectional modulators of EGFR functions in cancer cells. In addition, we will present the emerging evidence for microRNAs being specifically modulated by oncogenic EGFR mutants and we will discuss how this impinges on EGFRmut driven chemoresistance, which fits into the tumor heterogeneity-driven cancer progression. Finally, we discuss how other non-coding RNA species are emerging as important modulators of cancer progression and why the scenario depicted herein is destined to become increasingly complex in the future.

MicroRNA-128-3p-mediated depletion of Drosha promotes lung cancer cell migration.

Alteration in microRNAs (miRNAs) expression is a frequent finding in human cancers. In particular, widespread miRNAs down-regulation is a hallmark of malignant transformation. In the present report, we showed that the miR-128-3p, which is up-regulated in lung cancer tissues, has Drosha and Dicer, two key enzymes of miRNAs processing, as the main modulation targets leading to the widespread down-regulation of miRNA expression. We observed that the miRNAs downregulation induced by miR-128-3p contributed to the tumorigenic properties of lung cancer cells. In particular, miR-128-3p-mediated miRNAs down-regulation contributed to aberrant SNAIL and ZEB1 expression thereby promoting the epithelial-to-mesenchymal transition (EMT) program. Drosha also resulted to be implicated in the control of migratory phenotype as its expression counteracted miR-128-3p functional effects. Our study provides mechanistic insights into the function of miR-128-3p as a key regulator of the malignant phenotype of lung cancer cells. This also enforces the remarkable impact of Drosha and Dicer alteration in cancer, and in particular it highlights a role for Drosha in non-small-cell lung cancer cells migration.

Receptor-type protein-tyrosine phosphatase ζ is a functional receptor for interleukin-34.

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

Mapping Non-Coding RNAs in Space and Time: Another Weapon to Dissect Intra-Tumor Heterogeneity in Cancer Progression.

It is increasingly clear that Intratumor heterogeneity (ITH) fuels tumor evolution, matching the concept of cancer as a heterogeneous ecosystem of spatially and temporally modulated cell subpopulations, which exploits dynamic strategies to hijack local and systemic resources and tissue(s) space [...].

Harnessing the value of TCTP in breast cancer treatment resistance: an opportunity for personalized therapy.

Early identification of breast cancer (BC) patients at a high risk of progression may aid in therapeutic and prognostic aims. This is especially true for metastatic disease, which is responsible for most cancer-related deaths. Growing evidence indicates that the translationally controlled tumor protein (TCTP) may be a clinically relevant marker for identifying poorly differentiated aggressive BC tumors. TCTP is an intriguing protein with pleiotropic functions, which is involved in multiple signaling pathways. TCTP may also be involved in stress response, cell growth and proliferation-related processes, underlying its potential role in the initiation of metastatic growth. Thus, TCTP marks specific cancer cell sub-populations with pronounced stress adaptation, stem-like and immune-evasive properties. Therefore, we have shown that in vivo phospho-TCTP levels correlate with the response of BC cells to anti-HER2 agents. In this review, we discuss the clinical relevance of TCTP for personalized therapy, specific TCTP-targeting strategies, and currently available therapeutic agents. We propose TCTP as an actionable clinically relevant target that could potentially improve patient outcomes.

Interrogating colorectal cancer metastasis to liver: a search for clinically viable compounds and mechanistic insights in colorectal cancer Patient Derived Organoids.

BACKGROUND: Approximately 20-50% of patients presenting with localized colorectal cancer progress to stage IV metastatic disease (mCRC) following initial treatment and this is a major prognostic determinant. Here, we have interrogated a heterogeneous set of primary colorectal cancer (CRC), liver CRC metastases and adjacent liver tissue to identify molecular determinants of the colon to liver spreading. Screening Food and Drug Administration (FDA) approved drugs for their ability to interfere with an identified colon to liver metastasis signature may help filling an unmet therapeutic need. METHODS: RNA sequencing of primary colorectal cancer specimens vs adjacent liver tissue vs synchronous and asynchronous liver metastases. Pathways enrichment analyses. The Library of Integrated Network-based Cellular Signatures (LINCS)-based and Connectivity Map (CMAP)-mediated identification of FDA-approved compounds capable to interfere with a 22 gene signature from primary CRC and liver metastases. Testing the identified compounds on CRC-Patient Derived Organoid (PDO) cultures. Microscopy and Fluorescence Activated Cell Sorting (FACS) based analysis of the treated PDOs. RESULTS: We have found that liver metastases acquire features of the adjacent liver tissue while partially losing those of the primary tumors they derived from. We have identified a 22-gene signature differentially expressed among primary tumors and metastases and validated in public databases. A pharmacogenomic screening for FDA-approved compounds capable of interfering with this signature has been performed. We have validated some of the identified representative compounds in CRC-Patient Derived Organoid cultures (PDOs) and found that pentoxyfilline and, to a minor extent, dexketoprofen and desloratadine, can variably interfere with number, size and viability of the CRC -PDOs in a patient-specific way. We explored the pentoxifylline mechanism of action and found that pentoxifylline treatment attenuated the 5-FU elicited increase of ALDHhigh cells by attenuating the IL-6 mediated STAT3 (tyr705) phosphorylation. CONCLUSIONS: Pentoxifylline synergizes with 5-Fluorouracil (5-FU) in attenuating organoid formation. It does so by interfering with an IL-6-STAT3 axis leading to the emergence of chemoresistant ALDHhigh cell subpopulations in 5-FU treated PDOs. A larger cohort of CRC-PDOs will be required to validate and expand on the findings of this proof-of-concept study.

Butein-instigated miR-186-5p-dependent modulation of TWIST1 affects resistance to cisplatin and bioenergetics of Malignant Pleural Mesothelioma cells.

Aim: Malignant pleural mesothelioma is a chemoresistant tumor, and biphasic and sarcomatoid histologies portend the worst prognosis for malignant pleural mesothelioma (MPM) patients. We obtained the microRNA expression profile of three biphasic-sarcomatoid MPM cell lines to identify commonly expressed microRNAs and evaluate the effect of butein, a chemo-sensitizing compound, on this microRNA subset. Methods: Nanostring-based microRNA profiling and analysis through the ROSALIND platform were employed to identify the commonly modulated microRNAs and their targets. MicroRNA-mimic transfection, Luciferase assay, and Western blotting were employed to show specific perturbation of TWIST1 levels by miR-186-5p. Sphere-forming assays, invasion assay, and metabolic profiling were used to assess the biological consequences of the butein-instigated miR-186-5p-mediated perturbation of TWIST1 levels. TGCA analysis was used to search for the correlation between TWIST1 and miR-186-5p levels in biphasic and epithelioid MPM specimens. Results: We identified a set of perturbed microRNAs, common to three biphasic/sarcomatoid MPM cell lines, after butein treatment. When focusing on miR-186-5p, we unraveled a butein-ignited and miR-186-5p-mediated modulation of TWIST1 levels which affected the 3D anchorage-independent growth, cisplatin resistance, invasion, and bioenergetics of the MPM cell lines tested. We showed that miR-186-5p and TWIST1 levels are anti-correlated in biphasic MPM specimens from TCGA. Conclusion: We unraveled a novel mechanism of action of butein, which attenuated the pro-tumorigenic features of MPM at least through a miR-186-5p-TWIST1 axis. We suggest that those activities converge into the chemo-sensitizing effect of this compound and may be of translational relevance.

Breast cancer metastasis: Is it a matter of OMICS and proper ex-vivo models?

Genomics has greatly increased the understanding of the study of breast cancer (BC) and has shaped the concept of intra-tumor heterogeneity, currently recognized as a propelling force for cancer progression. In this context, knowledge and understanding of metastatic breast cancer (mBC) has somehow lagged behind that of primary breast cancer. This may be explained by the relative scarcity of matched mBC samples, however it is possible that the mutation spectrum obtained from primary BC does not capture the full complexity of the metastatic disease. Here, we provide a few examples supporting this possibility, from public databases. We evoke the need to perform an integrated multi-OMICS characterization of mBC, to obtain a broad understanding of this complex disease, whose evolution cannot be explained solely by genomics. Pertinent to this, we suggest that rather an infrequent use of Patient-Derived -Tumor-Organoids (PDTOs) may be influenced by assuming that the metastatic conditions of PDTOs growth (mPDTOs) should be similar to those of the tissue of origin. We challenge this view by suggesting that the use of "target-organ inspired" growth conditions for mPDTOs, may better fit the emerging knowledge of metastatic disease. Thus, the integrated use of multi-OMICS and of clinically relevant mPDTOs may allow a further understanding of such disease and foster therapeutically relevant advances. We believe that our points may be valid for other solid cancers.

UV-induced fragmentation of Cajal bodies.

The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28gamma (proteasome activator subunit gamma). The presence of PA28gamma in coilin-containing complexes is increased by UV-C. Overexpression of PA28gamma, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference-mediated knockdown of PA28gamma attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28gamma as a novel regulator of CB integrity.

EphA2 and EGFR: Friends in Life, Partners in Crime. Can EphA2 Be a Predictive Biomarker of Response to Anti-EGFR Agents?

The Eph receptors represent the largest group among Receptor Tyrosine kinase (RTK) families. The Eph/ephrin signaling axis plays center stage during development, and the deep perturbation of signaling consequent to its dysregulation in cancer reveals the multiplicity and complexity underlying its function. In the last decades, they have emerged as key players in solid tumors, including colorectal cancer (CRC); however, what causes EphA2 to switch between tumor-suppressive and tumor-promoting function is still an active theater of investigation. This review summarizes the recent advances in understanding EphA2 function in cancer, with detail on the molecular determinants of the oncogene-tumor suppressor switch function of EphA2. We describe tumor context-specific examples of EphA2 signaling and the emerging role EphA2 plays in supporting cancer-stem-cell-like populations and overcoming therapy-induced stress. In such a frame, we detail the interaction of the EphA2 and EGFR pathway in solid tumors, including colorectal cancer. We discuss the contribution of the EphA2 oncogenic signaling to the resistance to EGFR blocking agents, including cetuximab and TKIs.

Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma.

Background High resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed. METHODS: Mass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells. RESULTS: We first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug. CONCLUSIONS: AA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

Metformin: Metabolic Rewiring Faces Tumor Heterogeneity.

Tumor heterogeneity impinges on all the aspects of tumor history, from onset to metastasis and relapse. It is growingly recognized as a propelling force for tumor adaptation to environmental and micro-environmental cues. Metabolic heterogeneity perfectly falls into this process. It strongly contributes to the metabolic plasticity which characterizes cancer cell subpopulations-capable of adaptive switching under stress conditions, between aerobic glycolysis and oxidative phosphorylation-in both a convergent and divergent modality. The mitochondria appear at center-stage in this adaptive process and thus, targeting mitochondria in cancer may prove of therapeutic value. Metformin is the oldest and most used anti-diabetic medication and its relationship with cancer has witnessed rises and falls in the last 30 years. We believe it is useful to revisit the main mechanisms of action of metformin in light of the emerging views on tumor heterogeneity. We first analyze the most consolidated view of its mitochondrial mechanism of action and then we frame the latter in the context of tumor adaptive strategies, cancer stem cell selection, metabolic zonation of tumors and the tumor microenvironment. This may provide a more critical point of view and, to some extent, may help to shed light on some of the controversial evidence for metformin's anticancer action.

Che-1/AATF-induced transcriptionally active chromatin promotes cell proliferation in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of "active chromatin" by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome.

Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDHbright cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.

MiR-204 down-regulation elicited perturbation of a gene target signature common to human cholangiocarcinoma and gastric cancer.

BACKGROUND & AIMS: There is high need of novel diagnostic and prognostic tools for tumors of the digestive system, such as gastric cancer and cholangiocarcinoma. We recently found that miR-204 was deeply downregulated in gastric cancer tissues. Here we investigated whether this was common to other tumors of the digestive system and whether this elicited a miR-204-dependent gene target signature, diagnostically and therapeutically relevant. Finally, we assessed the contribution of the identified target genes to the cell cycle progression and clonogenicity of gastric cancer and cholangiocarcinoma cell lines. METHODS: We employed quantitative PCR and Affymetrix profiling for gene expression studies. In silico analysis aided us to identifying a miR-204 target signature in publicly available databases (TGCA). We employed transient transfection experiments, clonogenic assays and cell cycle profiling to evaluate the biological consequences of miR-204 perturbation. RESULTS: We identified a novel miR-204 gene target signature perturbed in gastric cancer and in cholangiocarcinoma specimens. We validated its prognostic relevance and mechanistically addressed its biological relevance in GC and CC cell lines. CONCLUSIONS: We suggest that restoring the physiological levels of miR-204 in some gastrointestinal cancers might be exploited therapeutically.