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Epidermal growth factor receptor (EGFR) is a well-exploited therapeutic target in metastatic colorectal cancer (mCRC). Unfortunately, not all patients benefit from current EGFR inhibitors. Mass spectrometry–based proteomics and phosphoproteomics were performed on 30 genomically and pharmacologically characterized mCRC patient–derived xenografts (PDXs) to investigate the molecular basis of response to EGFR blockade and identify alternative drug targets to overcome resistance. Both the tyrosine and global phosphoproteome as well as the proteome harbored distinctive response signatures. We found that increased pathway activity related to mitogen-activated protein kinase (MAPK) inhibition and abundant tyrosine phosphorylation of cell junction proteins, such as CXADR and CLDN1/3, in sensitive tumors, whereas epithelial-mesenchymal transition and increased MAPK and AKT signaling were more prevalent in resistant tumors. Furthermore, the ranking of kinase activities in single samples confirmed the driver activity of ERBB2, EGFR, and MET in cetuximab-resistant tumors. This analysis also revealed high kinase activity of several members of the Src and ephrin kinase family in 2 CRC PDX models with genomically unexplained resistance. Inhibition of these hyperactive kinases, alone or in combination with cetuximab, resulted in growth inhibition of ex vivo PDX-derived organoids and in vivo PDXs. Together, these findings highlight the potential value of phosphoproteomics to improve our understanding of anti-EGFR treatment and response prediction in mCRC and bring to the forefront alternative drug targets in cetuximab-resistant tumors. Editor's summary: Epidermal growth factor receptor (EGFR)–targeted therapy is a common treatment for patients with metastatic colorectal cancer (mCRC); however, some patients fail to respond. Here, Beekhof et al. performed phosphoproteomics on a cohort of 30 patient-derived xenografts to identify non-genomic mechanisms of resistance and sensitivity. The authors found increased kinase activity in resistant tumors, including two models with unexplained genomic resistance. Inhibition of these kinases overcame resistance, highlighting the use of phosphoproteomics in identifying new drug targets for patients with mCRC. —Dorothy Hallberg [ABSTRACT FROM AUTHOR]

In colorectal cancer, the mechanisms underlying tumor aggressiveness require further elucidation. Taking advantage of a large panel of human metastatic colorectal cancer xenografts and matched stem‐like cell cultures (m‐colospheres), here we show that the overexpression of microRNA 483‐3p (miRNA‐483‐3p; also known as MIR‐483‐3p), encoded by a frequently amplified gene locus, confers an aggressive phenotype. In m‐colospheres, endogenous or ectopic miRNA‐483‐3p overexpression increased proliferative response, invasiveness, stem cell frequency, and resistance to differentiation. Transcriptomic analyses and functional validation found that miRNA‐483‐3p directly targets NDRG1, known as a metastasis suppressor involved in EGFR family downregulation. Mechanistically, miRNA‐483‐3p overexpression induced the signaling pathway triggered by ERBB3, including AKT and GSK3β, and led to the activation of transcription factors regulating epithelial–mesenchymal transition (EMT). Consistently, treatment with selective anti‐ERBB3 antibodies counteracted the invasive growth of miRNA‐483‐3p‐overexpressing m‐colospheres. In human colorectal tumors, miRNA‐483‐3p expression inversely correlated with NDRG1 and directly correlated with EMT transcription factor expression and poor prognosis. These results unveil a previously unrecognized link between miRNA‐483‐3p, NDRG1, and ERBB3‐AKT signaling that can directly support colorectal cancer invasion and is amenable to therapeutic targeting. [ABSTRACT FROM AUTHOR]

Motivation The transition from evaluating a single time point to examining the entire dynamic evolution of a system is possible only in the presence of the proper framework. The strong variability of dynamic evolution makes the definition of an explanatory procedure for data fitting and clustering challenging. Results We developed CONNECTOR, a data-driven framework able to analyze and inspect longitudinal data in a straightforward and revealing way. When used to analyze tumor growth kinetics over time in 1599 patient-derived xenograft growth curves from ovarian and colorectal cancers, CONNECTOR allowed the aggregation of time-series data through an unsupervised approach in informative clusters. We give a new perspective of mechanism interpretation, specifically, we define novel model aggregations and we identify unanticipated molecular associations with response to clinically approved therapies. Availability and implementation CONNECTOR is freely available under GNU GPL license at https://qbioturin.github.io/connector and https://doi.org/10.17504/protocols.io.8epv56e74g1b/v1. [ABSTRACT FROM AUTHOR]

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab [...]

Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1-associated ErbB2/Neu oncogenic kinase. In sum, Sema3E-Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis., Introduction Semaphorins are secreted and membrane-bound signals, originally implicated in the control of axonal wiring (1). They comprise a wide protein family and are involved in a range of functions, [...]

Despite being a cell-matrix adhesion molecule, [beta]4 integrin can prompt the multiplication of neoplastic cells dislodged from their substrates (anchorage-independent growth). However, the molecular events underlying this atypical behavior remain partly unexplored. We found that activation of the Met receptor for hepatocyte growth factor results in the tyrosine phosphorylation of [beta]4, which is instrumental for integrin-mediated recruitment of the tyrosine phosphatase Shp2. Shp2 binding to [beta]4 enhances the activation of Src, which, in turn, phosphorylates the multiadaptor Gab1 predominantly on consensus sites for Grb2 association, leading to privileged stimulation of the Ras-extracellular signal-regulated kinase (ERK) cascade. This signaling axis can be inhibited by small interfering RNA-mediated [beta]4 depletion, by a [beta]4 mutant unable to bind Shp2, and by pharmacological and genetic inhibition of Shp2 or Src. Preservation of the [beta]4 docking sites for Shp2 as well as the integrity of Shp2, Src, or ERK activity are required for the [beta]4-mediated induction of anchorage-independent growth. These results unravel a novel pathway whereby [beta]4 directs tyrosine kinase-based signals toward adhesion-unrelated outcomes.

Author(s): Paolo M Comoglio [1]; Livio Trusolino [1] The extracellular matrix (ECM) is a complex molecular milieu that provides cells with the tensile scaffold necessary for appropriate assembly into three-dimensional [...]

Research shows that tyrosine phosphorylated alpha6beta4 integrin combines with adapter protein Shc and P13K leading to hepatocyte growth factor-triggered activation of Ras- and P13K-dependent pathways. Data suggest that the integrin helps cancer invasion by amplifying biochemical responses.

Research was conducted to demonstrate that the alpha-v-beta-3 integrin is expressed by normal cells in a latent state characterized by its inability to form cytoskeletal connections and to promote cell adhesion to extracellular matrix ligands. A cellular system of human thyroid clones was used to investigate the influence of cytokines on nonfunctional epithelial integrins. Results indicate the importance of cytokine-based autocrine loops for the physiological regulation of integrin activation.

Abstract Background Gastric cancer is the second leading cause of cancer mortality in the world. The receptor tyrosine kinase MET is constitutively activated in many gastric cancers and its expression is strictly required for survival of some gastric cancer cells. Thus, MET is considered a good candidate for targeted therapeutic intervention in this type of tumor, and MET inhibitors recently entered clinical trials. One of the major problems of therapies targeting tyrosine kinases is that many tumors are not responsive to treatment or eventually develop resistance to the drugs. Perspective studies are thus mandatory to identify the molecular mechanisms that could cause resistance to these therapies. Results Our in vitro and in vivo results demonstrate that, in MET-addicted gastric cancer cells, the activation of HER (Human Epidermal Receptor) family members induces resistance to MET silencing or inhibition by PHA-665752 (a selective kinase inhibitor). We provide molecular evidences highlighting the role of EGFR, HER3, and downstream signaling pathways common to MET and HER family in resistance to MET inhibitors. Moreover, we show that an in vitro generated gastric cancer cell line resistant to MET-inhibition displays overexpression of HER family members, whose activation contributes to maintenance of resistance. Conclusions Our findings predict that gastric cancer tumors bearing constitutive activation of HER family members are poorly responsive to MET inhibition, even if this receptor is constitutively active. Moreover, the appearance of these alterations might also be responsible for the onset of resistance in initially responsive tumors.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild‐type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse‐phase protein array‐based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild‐type metastatic CRC tumours. Responses of tumours to anti‐EGFR therapy with cetuximab were recorded in patient‐derived xenograft (PDX) models. Unsupervised hierarchical clustering of pretreatment tumour tissue identified three clusters, of which Cluster C3 was exclusively composed of responders. Clusters C1 and C2 exhibited mixed responses. None of the three protein clusters exhibited a significant correlation with transcriptome‐based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab‐sensitive and cetuximab‐resistant tumours: PDK1 (S241), caspase‐8, Shc (Y317), Stat3 (Y705), p27, GSK‐3β (S9), HER3, PKC‐α (S657), EGFR (Y1068), Akt (S473), S6 ribosomal protein (S240/244), HER3 (Y1289), NF‐κB‐p65 (S536) and Gab‐1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo‐)protein analysis of matched pretreated and posttreated models furthermore showed significant reduction of Gab‐1 (Y627) and GSK‐3β (S9) exclusively in responding models, suggesting novel targets for treatment. What's new? Therapies that neutralize epidermal growth factor receptor (EGFR) activity have greatly improved survival in metastatic colorectal cancer (mCRC) patients. Still, many patients will fail to respond to therapy, despite having wild‐type KRAS, which generally predicts anti‐EGFR therapeutic efficacy. Here, statistical analysis of reverse phase protein array data from KRAS, BRAF, NRAS, and PI3KCA wild‐type mCRC patient‐derived xenograft mouse models shows that response to the anti‐EGFR therapy cetuximab is reliably predicted by a 14 (phospho)protein marker signature. The findings further suggest that GSK‐3β, dysregulation of which is associated with cancer, is a promising target for co‐treatment with cetuximab. [ABSTRACT FROM AUTHOR]

Background: The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing.Methods: We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided.Results: Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models ( P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma.Conclusions: These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC. [ABSTRACT FROM AUTHOR]

The approval in July of a law on animal experimentation in Italy could irreversibly jeopardize the country's medical research, particularly in drug development, and exclude Italy's scientists from international funding [...]

The article discusses the study on the sensitivity of anti-epidermal growth factor receptor (EGFR) extracellular domain (ECD)-resistant variants of colorectal cancers to EGFR blockade by the oligoclonal antibody MM-151. MM-151 reportedly binds various regions of EGFR ECD which inhibit EGFT signalling and cell growth. The results revealed that MM-151 can overcome resistance to cetuximab and panitumumab in colon cancer.

Background: Tankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front. Results: Here we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation. Conclusions: Collectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells. [ABSTRACT FROM AUTHOR]

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component. [ABSTRACT FROM AUTHOR]

The article looks at a study regarding insulin-like growth factor 2 (IGF2) which identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-epidermal growth factor receptor (EGFR) therapies. It mentions therapies tailored around hypothesis-generating molecular features in patient-derived xenografts and resistance-conferring mutations. The study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker.

Background Standard chemotherapy in unresectable biliary tract carcinoma (BTC) patients is based on gemcitabine combined with platinum derivatives. However, primary or acquired resistance is inevitable and no second-line chemotherapy is demonstrated to be effective. Thus, there is an urgent need to identify new alternative (chemo)therapy approaches. Methods We evaluated the mechanism of action of ET-743 in preclinical models of BTC. Six BTC cell lines (TFK-1, EGI-1, TGBC1, WITT, KMCH, HUH28), two primary cell cultures derived from BTC patients, the EGI-1 and a new established BTC patient-derived xenografts, were used as preclinical models to investigate the anti-tumor activity of ET-743 in vitro and in vivo. Gene expression profiling was also analyzed upon ET-743 treatment in in vivo models. Results We found that ET-743 inhibited cell growth of BTC cell lines and primary cultures (IC50 ranging from 0.37 to 3.08 nM) preferentially inducing apoptosis and activation of the complex DNA damage-repair proteins (p-ATM, p-p53 and p-Histone H2A.x) in vitro. In EGI-1 and patient-derived xenografts, ET-743 induced tumor growth delay and reduction of vasculogenesis. In vivo ET-743 induced a deregulation of genes involved in cell adhesion, stress-related response, and in pathways involved in cholangiocarcinogenesis, such as the IL- 6, Sonic Hedgehog and Wnt signaling pathways. Conclusions These results suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials in BTC patients resistant to standard chemotherapy. [ABSTRACT FROM AUTHOR]

Only approximately 10 % of genetically unselected patients with chemorefractory metastatic colorectal cancer experience tumor regression when treated with the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab ('primary' or 'de novo' resistance). Moreover, nearly all patients whose tumors initially respond inevitably become refractory ('secondary' or 'acquired' resistance). An ever-increasing number of predictors of both primary and acquired resistance to anti-EGFR antibodies have been described, and it is now evident that most of the underlying mechanisms significantly overlap. By trying to extrapolate a unifying perspective out of many idiosyncratic details, here, we discuss the molecular underpinnings of therapeutic resistance, summarize research efforts aimed to improve patient selection, and present alternative therapeutic strategies that are now under development to increase response and combat relapse. [ABSTRACT FROM AUTHOR]

Summary: There are no effective therapies for the ∼30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment. [Copyright &y& Elsevier]