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.

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.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Synergistic interactions with PI3K inhibition that induce apoptosis.

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition.

RNF20 and histone H2B ubiquitylation exert opposing effects in Basal-Like versus luminal breast cancer.

Breast cancer subtypes display distinct biological traits that influence their clinical behavior and response to therapy. Recent studies have highlighted the importance of chromatin structure regulators in tumorigenesis. The RNF20-RNF40 E3 ubiquitin ligase complex monoubiquitylates histone H2B to generate H2Bub1, while the deubiquitinase (DUB) USP44 can remove this modification. We found that RNF20 and RNF40 expression and global H2Bub1 are relatively low, and USP44 expression is relatively high, in basal-like breast tumors compared with luminal tumors. Consistent with a tumor-suppressive role, silencing of RNF20 in basal-like breast cancer cells increased their proliferation and migration, and their tumorigenicity and metastatic capacity, partly through upregulation of inflammatory cytokines. In contrast, in luminal breast cancer cells, RNF20 silencing reduced proliferation, migration and tumorigenic and metastatic capacity, and compromised estrogen receptor transcriptional activity, indicating a tumor-promoting role. Notably, the effects of USP44 silencing on proliferation and migration in both cancer subtypes were opposite to those of RNF20 silencing. Hence, RNF20 and H2Bub1 have contrasting roles in distinct breast cancer subtypes, through differential regulation of key transcriptional programs underpinning the distinctive traits of each subtype.

An in-tumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive form of epithelial ovarian cancer, for which few targeted therapies exist. To search for new therapeutic target proteins, we performed an in vivo shRNA screen using an established human HGSOC cell line growing either subcutaneously or intraperitoneally in immunocompromised mice. We identified genes previously implicated in ovarian cancer such as AURKA1, ERBB3, CDK2, and mTOR, as well as several novel candidates including BRD4, VRK1, and GALK2. We confirmed, using both genetic and pharmacologic approaches, that the activity of BRD4, an epigenetic transcription modulator, is necessary for proliferation/survival of both an established human ovarian cancer cell line (OVCAR8) and a subset of primary serous ovarian cancer cell strains (DFs). Among the DFs tested, the strains sensitive to BRD4 inhibition revealed elevated expression of either MYCN or c-MYC, with MYCN expression correlating closely with JQ1 sensitivity. Accordingly, primary human xenografts derived from high-MYCN or c-MYC strains exhibited sensitivity to BRD4 inhibition. These data suggest that BRD4 inhibition represents a new therapeutic approach for MYC-overexpressing HGSOCs.

KRAS and YAP1 converge to regulate EMT and tumor survival.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.

Consistency test of the cell cycle: roles for p53 and EGR1.

Mammalian cells are constantly exposed to multiple mitogens and, hence, have developed machineries that help them ignore fortuitous signals. In a recent report in Molecular Cell, we highlighted the molecular details of such a noise-reduction filter, including roles for EGR1, AKT, and p53. Brief exposure to a mitogen drives formation of inhibitory p53-chromatin complexes, which are disabled only if the growth factor is still present several hours later. We propose that this "consistency test" prevents repeated division cycles of normal cells but might become defective in most cancer cells.

EGR1 and the ERK-ERF axis drive mammary cell migration in response to EGF.

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.

Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

EGF decreases the abundance of microRNAs that restrain oncogenic transcription factors.

Epidermal growth factor (EGF) stimulates cells by launching gene expression programs that are frequently deregulated in cancer. MicroRNAs, which attenuate gene expression by binding complementary regions in messenger RNAs, are broadly implicated in cancer. Using genome-wide approaches, we showed that EGF stimulation initiates a coordinated transcriptional program of microRNAs and transcription factors. The earliest event involved a decrease in the abundance of a subset of 23 microRNAs. This step permitted rapid induction of oncogenic transcription factors, such as c-FOS, encoded by immediate early genes. In line with roles as suppressors of EGF receptor (EGFR) signaling, we report that the abundance of this early subset of microRNAs is decreased in breast and in brain tumors driven by the EGFR or the closely related HER2. These findings identify specific microRNAs as attenuators of growth factor signaling and oncogenesis.

Systems biology of growth factor-induced receptor endocytosis.

Clathrin-mediated endocytosis sorts for degradation of more than 50 different growth factor receptors capable of relaying growth and differentiation signals by means of their cytoplasm-facing, intrinsic tyrosine kinase activity. The kinetics and alternative routings of receptor endocytosis critically regulate growth factor signaling, which underscores the importance of understanding mechanisms underlying fail-safe operation (robustness) and fidelity of the pathway. Like other robust systems, a layered hub-centric network controls receptor endocytosis. Characteristically, the modular hubs (e.g., AP2-Eps15 and Hrs) contain a membrane-anchoring lipid-binding domain, an ubiquitin-binding module, which recruits ubiquitinylated cargo, and a machinery enabling homo-assembly. Scheduled hub transitions, as well as cascades of Rab family guanosine triphosphatases and membrane bending machineries, define points of commitment to vesicle budding, thereby securing unidirectional trafficking. System's bistability permits stimulation by a growth factor, which oscillates a series of switches based on posttranslational protein modifications (i.e., phosphorylation, ubiquitinylation and neddylation), as well as transient low-affinity/high-avidity protein assemblies. Cbl family ubiquitin ligases, along with a set of phosphotyrosine-binding adaptors (e.g., Grb2), integrate receptor endocytosis into the densely wired networks of signal transduction pathways, which are involved in health and disease.

p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy.

The epidermal growth factor receptor (EGFR) frequently associates with cancer and already serves as a target for therapy. We report that inflammatory cytokines and ultraviolet (UV) irradiation respectively induce transient or sustained phosphorylation of EGFR. Subsequently, EGFR internalizes via a Clathrin-mediated process. In cytokine-stimulated cells, EGFR recycles back to the cell surface, whereas in irradiated cells it arrests in Rab5-containing endosomes. Under both conditions, receptor internalization is instigated by the p38 stress-induced kinase. The underlying mechanism entails phosphorylation of EGFR at a short segment (amino acids 1002-1022) containing multiple serines and threonines, as well as phosphorylation of two Rab5 effectors, EEA1 and GDI. Like UV irradiation, a chemotherapeutic agent activates p38 and accelerates receptor internalization. We demonstrate that abrogating EGFR internalization reduces the efficacy of chemotherapy-induced cell death. Hence, by preventing EGFR-mediated survival signaling, the internalization route we uncovered enhances the cytotoxic effect of drugs like cis-platinum, which may underlie interactions between chemotherapy and EGFR-targeting drugs.

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases.

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.

Phosphorylation of carboxyl-terminal tyrosines modulates the specificity of Sprouty-2 inhibition of different signaling pathways.

Sprouty proteins are evolutionarily conserved negative feedback regulators of multiple receptor tyrosine kinases. Mammalian versions of these proteins differentially regulate signaling induced by the fibroblast and the epidermal growth factors (FGF and EGF, respectively). Herein we show that, although both growth factors elevate expression of Sprouty-2, FGF- and not EGF-induced activation of the Erk/MAPK pathway is inhibited by Sprouty-2. Attenuation of FGF-signaling is accompanied by the induction of Sprouty-2 phosphorylation on the amino-terminal as well as carboxyl-terminal tyrosine residues, which are less effectively modified upon EGF treatment. Mutagenesis of carboxyl-terminal tyrosines, especially a newly identified phosphorylation site, tyrosine 227, impaired the inhibitory activity of Sprouty-2. These results attribute a novel role for carboxyl-terminal tyrosine residues and yet unidentified phosphotyrosine-binding proteins in the differential regulation of Sprouty-2 activity.

Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding.

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.

Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7.

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 to the receptor's tail. We show that all four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia.