3D Culture Models with CRISPR Screens Reveal Hyperactive NRF2 as a Prerequisite for Spheroid Formation via Regulation of Proliferation and Ferroptosis.

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

Synthetic Lethal and Resistance Interactions with BET Bromodomain Inhibitors in Triple-Negative Breast Cancer.

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Uncovering a tumor suppressor for triple-negative breast cancers.

"Triple-negative" breast cancers are aggressive malignancies that respond poorly to treatments. Now Sun et al. (2011) find that the activity of the protein tyrosine phosphatase PTPN12 is lost in a large percentage of this breast cancer subtype, offering molecular drivers and possible therapeutic targets for this heterogeneous and intractable cancer.

Tumor self-seeding: bidirectional flow of tumor cells.

Circulating tumor cells are responsible for seeding metastatic growth at distant sites. Kim et al. (2009) now discover that circulating tumor cells can reinfiltrate tumors at their primary organs and promote tumor progression.

Mutant p53 drives invasion by promoting integrin recycling.

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.

Corrigendum: Chaperone-mediated autophagy degrades mutant p53.

In the above-mentioned article, it has come to the authors' attention that, during the preparation of Figure 5C and Supplemental Figure S2C for the final version of this article, the authors unintentionally assembled incorrect tubulin immunoblots due to similarities in the markings or names, such as FLT3 versus FT, between two similar experiments. The amended versions of these figures are shown below. Neither the quantitative determinations nor the conclusions of this article are altered. The authors apologize for these errors.

Fibroblast-tumor cell signaling limits HER2 kinase therapy response via activation of MTOR and antiapoptotic pathways.

Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+ breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+ breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic-phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.

Role for polo-like kinase 4 in mediation of cytokinesis.

The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.

Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals.

The EGF-stimulated ERK/MAPK pathway is a key conduit for cellular proliferation signals and a therapeutic target in many cancers. Here, we characterize two central quantitative aspects of this pathway: the mechanism by which signal strength is encoded and the response curve relating signal output to proliferation. Under steady-state conditions, we find that ERK is activated in discrete, asynchronous pulses with frequency and duration determined by extracellular concentrations of EGF spanning the physiological range. In genetically identical sister cells, cell-to-cell variability in pulse dynamics influences the decision to enter S phase. While targeted inhibition of EGFR reduces the frequency of ERK activity pulses, inhibition of MEK reduces their amplitude. Continuous response curves measured in multiple cell lines reveal that proliferation is effectively silenced only when ERK pathway output falls below a threshold of ~10%, indicating that high-dose targeting of the pathway is necessary to achieve therapeutic efficacy.

Chaperone-mediated autophagy degrades mutant p53.

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells.

Navitoclax enhances the effectiveness of EGFR-targeted antibody-drug conjugates in PDX models of EGFR-expressing triple-negative breast cancer.

BACKGROUND: Targeted therapies for triple-negative breast cancer (TNBC) are limited; however, the epidermal growth factor receptor (EGFR) represents a potential target, as the majority of TNBC express EGFR. The purpose of these studies was to evaluate the effectiveness of two EGFR-targeted antibody-drug conjugates (ADC: ABT-414; ABBV-321) in combination with navitoclax, an antagonist of the anti-apoptotic BCL-2 and BCL-XL proteins, in order to assess the translational relevance of these combinations for TNBC. METHODS: The pre-clinical efficacy of combined treatments was evaluated in multiple patient-derived xenograft (PDX) models of TNBC. Microscopy-based dynamic BH3 profiling (DBP) was used to assess mitochondrial apoptotic signaling induced by navitoclax and/or ADC treatments, and the expression of EGFR and BCL-2/XL was analyzed in 46 triple-negative patient tumors. RESULTS: Treatment with navitoclax plus ABT-414 caused a significant reduction in tumor growth in five of seven PDXs and significant tumor regression in the highest EGFR-expressing PDX. Navitoclax plus ABBV-321, an EGFR-targeted ADC that displays more effective wild-type EGFR-targeting, elicited more significant tumor growth inhibition and regressions in the two highest EGFR-expressing models evaluated. The level of mitochondrial apoptotic signaling induced by single or combined drug treatments, as measured by DBP, correlated with the treatment responses observed in vivo. Lastly, the majority of triple-negative patient tumors were found to express EGFR and co-express BCL-XL and/or BCL-2. CONCLUSIONS: The dramatic tumor regressions achieved using combined agents in pre-clinical TNBC models underscore the abilities of BCL-2/XL antagonists to enhance the effectiveness of EGFR-targeted ADCs and highlight the clinical potential for usage of such targeted ADCs to alleviate toxicities associated with combinations of BCL-2/XL inhibitors and systemic chemotherapies.

The cell biology of cell-in-cell structures.

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.

Oncogene-like induction of cellular invasion from centrosome amplification.

Centrosome amplification has long been recognized as a feature of human tumours; however, its role in tumorigenesis remains unclear. Centrosome amplification is poorly tolerated by non-transformed cells and, in the absence of selection, extra centrosomes are spontaneously lost. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumours, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumour progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behaviour is similar to that induced by overexpression of the breast cancer oncogene ERBB2 (ref. 4) and indeed enhances invasiveness triggered by ERBB2. Our data indicate that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation.

Identification of cancer genes that are independent of dominant proliferation and lineage programs.

Large, multidimensional cancer datasets provide a resource that can be mined to identify candidate therapeutic targets for specific subgroups of tumors. Here, we analyzed human breast cancer data to identify transcriptional programs associated with tumors bearing specific genetic driver alterations. Using an unbiased approach, we identified thousands of genes whose expression was enriched in tumors with specific genetic alterations. However, expression of the vast majority of these genes was not enriched if associations were analyzed within individual breast tumor molecular subtypes, across multiple tumor types, or after gene expression was normalized to account for differences in proliferation or tumor lineage. Together with linear modeling results, these findings suggest that most transcriptional programs associated with specific genetic alterations in oncogenes and tumor suppressors are highly context-dependent and are predominantly linked to differences in proliferation programs between distinct breast cancer subtypes. We demonstrate that such proliferation-dependent gene expression dominates tumor transcriptional programs relative to matched normal tissues. However, we also identified a relatively small group of cancer-associated genes that are both proliferation- and lineage-independent. A subset of these genes are attractive candidate targets for combination therapy because they are essential in breast cancer cell lines, druggable, enriched in stem-like breast cancer cells, and resistant to chemotherapy-induced down-regulation.

Critical questions in ovarian cancer research and treatment: Report of an American Association for Cancer Research Special Conference.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation.

Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECM-attached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation.

Psidin, a conserved protein that regulates protrusion dynamics and cell migration.

Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.

Outgrowth of single oncogene-expressing cells from suppressive epithelial environments.

Tumorigenesis is a clonal evolution process that is initiated from single cells within otherwise histologically normal tissue. It is unclear how single, sporadic mutant cells that have sustained oncogenic alterations evolve within a tightly regulated tissue environment. Here we investigated the effects of inducing oncogene expression in single cells in organotypic mammary acini as a model to elucidate the processes by which oncogenic alterations initiate clonal progression from organized epithelial environments. Sporadic cells induced to overexpress oncogenes that specifically perturb cell-cycle checkpoints (for example, E7 from human papilloma virus 16, and cyclin D1), deregulate Myc transcription or activate AKT signalling remained quiescent within growth-arrested acini. By contrast, single cells that overexpress ERBB2 initiated a cellular cascade involving cell translocation from the epithelial layer, as well as luminal outgrowth that is characteristic of neoplastic progression in early-stage epithelial tumours. In addition, ERBB2-mediated cell translocation to the lumen was found to depend on extracellular-regulated kinase and matrix metalloproteinase activities, and genetic alterations that perturb local cell-matrix adhesion drove cell translocation. We also provide evidence that luminal cell translocation may drive clonal selection by promoting either the death or the expansion of quiescent oncogene-expressing cells, depending on whether the pre-existing alterations allow anchorage-independent survival and growth. Our data show that the initial outgrowth of single oncogene-expressing cells from organized epithelial structures is a highly regulated process, and we propose that a cell translocation mechanism allows sporadic mutant cells to evade suppressive micro-environments and elicits clonal selection for survival and proliferative expansion outside the native niches of these cells.