Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

PURPOSE: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. METHODS: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. RESULTS: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. CONCLUSION: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.

A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis.

The discrimination of protein biological functions in different phases of the cell cycle is limited by the lack of experimental approaches that do not require pre-treatment with compounds affecting the cell cycle progression. Therefore, potential cycle-specific biological functions of a protein of interest could be biased by the effects of cell treatments. The OsTIR1/auxin-inducible degron (AID) system allows "on demand" selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population, as with previously available approaches. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. We validated the ROLECCS technology by down regulating the protein levels of TP53, one of the most studied tumor suppressor genes, with a widely known role in cell cycle progression. By using our novel tool, we observed that TP53 degradation is associated with increased number of micronuclei, and this phenotype is specifically achieved when TP53 is lost in S/G2/M phases of the cell cycle, but not in G1. Therefore, we propose the use of the ROLECCS system as a new improved way of studying the differential roles that target proteins may have in specific phases of the cell cycle.

Live-Cell Sender-Receiver Co-cultures for Quantitative Measurement of Paracrine Signaling Dynamics, Gene Expression, and Drug Response.

Paracrine signaling is a fundamental process regulating tissue development, repair, and pathogenesis of diseases such as cancer. Herein we describe a method for quantitatively measuring paracrine signaling dynamics, and resultant gene expression changes, in living cells using genetically encoded signaling reporters and fluorescently tagged gene loci. We discuss considerations for selecting paracrine "sender-receiver" cell pairs, appropriate reporters, the use of this system to ask diverse experimental questions and screen drugs blocking intracellular communication, data collection, and the use of computational approaches to model and interpret these experiments.

BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis.

Cancer therapies trigger diverse cellular responses, ranging from apoptotic death to acquisition of persistent therapy-refractory states such as senescence. Tipping the balance toward apoptosis could improve treatment outcomes regardless of therapeutic agent or malignancy. We find that inhibition of the mitochondrial protein BCL-xL increases the propensity of cancer cells to die after treatment with a broad array of oncology drugs, including mitotic inhibitors and chemotherapy. Functional precision oncology and omics analyses suggest that BCL-xL inhibition redirects the outcome of p53 transcriptional response from senescence to apoptosis, which likely occurs via caspase-dependent down-modulation of p21 and downstream cytostatic proteins. Consequently, addition of a BCL-2/xL inhibitor strongly improves melanoma response to the senescence-inducing drug targeting mitotic kinase Aurora kinase A (AURKA) in mice and patient-derived organoids. This study shows a crosstalk between the mitochondrial apoptotic pathway and cell cycle regulation that can be targeted to augment therapeutic efficacy in cancers with wild-type p53.

JARID1-targeted histone H3 demethylase inhibitors exhibit anti-proliferative activity and overcome cisplatin resistance in canine oral melanoma cell lines.

Histone demethylases are overexpressed or display altered activity in numerous human cancers leading to alterations in cell cycle dynamics, DNA repair kinetics, and therapeutic resistance. Consequently, therapeutic targeting of histone demethylases has become an active and promising area of research in human oncology. However, the role of histone demethylases and the potential efficacy of demethylase inhibition in canine cancers remains largely unknown. In the present work, we addressed this knowledge gap by exploring the therapeutic potential of histone demethylase inhibitors (HDIs) in canine oral melanoma. Using canine melanoma cell lines, we determined that broad spectrum HDIs result in decreased cell survival and prolonged DNA damage repair kinetics. We then showed that JARID1B, a histone H3 demethylase implicated in proliferation-dormancy regulation and drug sensitivity in human cancers, is highly expressed in canine tumour tissues. HDIs targeting JARID1B, and related JARID1 family members, significantly reduced survival fractions in canine melanoma cell lines, but did not appear to modulate DNA damage repair kinetics like broad spectrum HDI treatments. Importantly, we found that the anti-proliferative effects of JARID1-targeted HDIs are preserved in cell lines resistant to platinum-based chemotherapeutics, suggesting that HDIs may serve as a viable therapeutic strategy when faced with oral melanomas that progress despite the use of conventional therapies.

The selective inhibitor of nuclear export (SINE) verdinexor exhibits biologic activity against canine osteosarcoma cell lines.

Verdinexor (KPT-335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose-dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21 to 74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50  = 21 µM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Systems-Level Properties of EGFR-RAS-ERK Signaling Amplify Local Signals to Generate Dynamic Gene Expression Heterogeneity.

Intratumoral heterogeneity is associated with aggressive tumor behavior, therapy resistance, and poor patient outcomes. Such heterogeneity is thought to be dynamic, shifting over periods of minutes to hours in response to signaling inputs from the tumor microenvironment. However, models of this process have been inferred from indirect or post-hoc measurements of cell state, leaving the temporal details of signaling-driven heterogeneity undefined. Here, we developed a live-cell model system in which microenvironment-driven signaling dynamics can be directly observed and linked to variation in gene expression. Our analysis reveals that paracrine signaling between two cell types is sufficient to drive continual diversification of gene expression programs. This diversification emerges from systems-level properties of the EGFR-RAS-ERK signaling cascade, including intracellular amplification of amphiregulin-mediated paracrine signals and differential kinetic filtering by target genes including Fra-1, c-Myc, and Egr1. Our data enable more precise modeling of paracrine-driven transcriptional variation as a generator of gene expression heterogeneity. A record of this paper's transparent peer review process is included in the Supplemental Information.

Experimental and engineering approaches to intracellular communication.

Communication between and within cells is essential for multicellular life. While intracellular signal transduction pathways are often specified in molecular terms, the information content they transmit remains poorly defined. Here, we review research efforts to merge biological experimentation with concepts of communication that emerge from the engineering disciplines of signal processing and control theory. We discuss the challenges of performing experiments that quantitate information transfer at the molecular level, and we highlight recent studies that have advanced toward a clearer definition of the information content carried by signaling molecules. Across these studies, we emphasize a theme of increasingly well-matched experimental and theoretical approaches to decode the data streams directing cellular behavior.

Microenvironmental Signals and Biochemical Information Processing: Cooperative Determinants of Intratumoral Plasticity and Heterogeneity.

Intra-tumor cellular heterogeneity is a major challenge in cancer therapy. Tumors are composed of multiple phenotypic subpopulations that vary in their ability to initiate metastatic tumors and in their sensitivity to chemotherapy. In many cases, cells can transition between these subpopulations, not by genetic mutation, but instead through reversible changes in signal transduction or gene expression programs. This plasticity begins at the level of the microenvironment where local autocrine and paracrine signals, exosomes, tumor-stroma interactions, and extracellular matrix (ECM) composition create a signaling landscape that varies over space and time. The integration of this complex array of signals engages signaling pathways that control gene expression. The resulting modulation of gene expression programs causes individual cells to sample a wide array of phenotypic states that support tumor growth, dissemination, and therapeutic resistance. In this review, we discuss how information flows dynamically within the microenvironmental landscape to inform cell state decisions and to create intra-tumoral heterogeneity. We address the role of plasticity in the acquisition of transient and prolonged drug resistant states and discuss how targeted pharmacological modification of the signaling landscape may be able to constrain phenotypic plasticity, leading to improved treatment responses.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Adenomatous polyposis coli mutants dominantly activate Hsf1-dependent cell stress pathways through inhibition of microtubule dynamics.

Cancer cells up-regulate cell stress pathways, including the protein chaperone Hsp90. Increases in Hsp90 are believed "buffer" mutant protein activities necessary for cancer phenotypes. Activation of the cell stress pathway also alters the transcriptional landscape of cells in ways that are critical for cancer progression. However, it is unclear when and how the cell stress pathway is de-regulated during cancer progression. Here we report that mutations in adenomatous polyposis coli (APC) found in colorectal cancer activate cell stress pathways in mouse intestinal crypt cells, prior to loss of heterozygosity at APC or to the appearance of canonical intestinal cancer markers. Hsp90 levels are elevated in normal APC heterozygote crypt cells and further elevated in non-cancer cells adjacent to dysplasias, suggesting that the Hsp90 stress pathway marks the "cancer-field" effect. Expression of mutant APC in normal human epithelial cells is sufficient to activate a cell stress pathway via perturbations in microtubule dynamics. Inhibition of microtubule dynamics is sufficient to activate an Hsf1-dependent increase in gene transcription and protein levels. We suggest that the early activation of this Hsf1 dependent cell stress pathway by mono-allelic mutations in APC can affect cell programming in a way that contributes to cancer onset.

Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore-microtubule binding sites.

The formation of functional kinetochores requires the accurate assembly of a large number of protein complexes. The Hsp90-Sgt1 chaperone complex is important for this process; however, its targets are not conserved and its exact contribution to kinetochore assembly is unclear. Here, we show that human Hsp90-Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites. Interestingly, coinhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less-robust microtubule-binding sites. We propose that a balance of Mis12 complex assembly and turnover is required for the efficient and accurate assembly of kinetochore-microtubule binding sites. These findings support a novel role for Hsp90-Sgt1 chaperones in ensuring the fidelity of multiprotein complex assembly.