Chemotherapy and the Extra-Tumor Immune Microenvironment: EXTRA-TIME.

SUMMARY: Understandably, conventional therapeutic strategies have focused on controlling primary tumors. We ask whether the cost of such strategies is actually an increased likelihood of metastatic relapse.

Multi-modal transcriptomic analysis unravels enrichment of hybrid epithelial/mesenchymal state and enhanced phenotypic heterogeneity in basal breast cancer.

Intra-tumoral phenotypic heterogeneity promotes tumor relapse and therapeutic resistance and remains an unsolved clinical challenge. It manifests along multiple phenotypic axes and decoding the interconnections among these different axes is crucial to understand its molecular origins and to develop novel therapeutic strategies to control it. Here, we use multi-modal transcriptomic data analysis - bulk, single-cell and spatial transcriptomics - from breast cancer cell lines and primary tumor samples, to identify associations between epithelial-mesenchymal transition (EMT) and luminal-basal plasticity - two key processes that enable heterogeneity. We show that luminal breast cancer strongly associates with an epithelial cell state, but basal breast cancer is associated with hybrid epithelial/mesenchymal phenotype(s) and higher phenotypic heterogeneity. These patterns were inherent in methylation profiles, suggesting an epigenetic crosstalk between EMT and lineage plasticity in breast cancer. Mathematical modelling of core underlying gene regulatory networks representative of the crosstalk between the luminal-basal and epithelial-mesenchymal axes recapitulate and thus elucidate mechanistic underpinnings of the observed associations from transcriptomic data. Our systems-based approach integrating multi-modal data analysis with mechanism-based modeling offers a predictive framework to characterize intra-tumor heterogeneity and to identify possible interventions to restrict it.

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

Single-cell multi-modal GAN reveals spatial patterns in single-cell data from triple-negative breast cancer.

Exciting advances in technologies to measure biological systems are currently at the forefront of research. The ability to gather data along an increasing number of omic dimensions has created a need for tools to analyze all of this information together, rather than siloing each technology into separate analysis pipelines. To advance this goal, we introduce a framework called the single-cell multi-modal generative adversarial network (scMMGAN) that integrates data from multiple modalities into a unified representation in the ambient data space for downstream analysis using a combination of adversarial learning and data geometry techniques. The framework's key improvement is an additional diffusion geometry loss with a new kernel that constrains the otherwise over-parameterized GAN. We demonstrate scMMGAN's ability to produce more meaningful alignments than alternative methods on a wide variety of data modalities and that its output can be used to draw conclusions from real-world biological experimental data.

Mapping Phenotypic Plasticity upon the Cancer Cell State Landscape Using Manifold Learning.

ABSTRACT: Phenotypic plasticity describes the ability of cancer cells to undergo dynamic, nongenetic cell state changes that amplify cancer heterogeneity to promote metastasis and therapy evasion. Thus, cancer cells occupy a continuous spectrum of phenotypic states connected by trajectories defining dynamic transitions upon a cancer cell state landscape. With technologies proliferating to systematically record molecular mechanisms at single-cell resolution, we illuminate manifold learning techniques as emerging computational tools to effectively model cell state dynamics in a way that mimics our understanding of the cell state landscape. We anticipate that "state-gating" therapies targeting phenotypic plasticity will limit cancer heterogeneity, metastasis, and therapy resistance. SIGNIFICANCE: Nongenetic mechanisms underlying phenotypic plasticity have emerged as significant drivers of tumor heterogeneity, metastasis, and therapy resistance. Herein, we discuss new experimental and computational techniques to define phenotypic plasticity as a scaffold to guide accelerated progress in uncovering new vulnerabilities for therapeutic exploitation.

The Complexities of Metastasis.

Therapies that prevent metastatic dissemination and tumor growth in secondary organs are severely lacking. A better understanding of the mechanisms that drive metastasis will lead to improved therapies that increase patient survival. Within a tumor, cancer cells are equipped with different phenotypic and functional capacities that can impact their ability to complete the metastatic cascade. That phenotypic heterogeneity can be derived from a combination of factors, in which the genetic make-up, interaction with the environment, and ability of cells to adapt to evolving microenvironments and mechanical forces play a major role. In this review, we discuss the specific properties of those cancer cell subgroups and the mechanisms that confer or restrict their capacity to metastasize.

CD44 Orchestrates Metastatic Teamwork.

Inhibition of metastatic cancer cell colonization and outgrowth is arguably one of the greatest therapeutic challenges. Reporting in Cancer Discovery, Liu et al. (2018) describe how homophilic interactions of CD44, a classical breast cancer stem cell marker, drive tumor cell aggregation outside the primary tumor to augment their metastatic potential.

IL-1ß inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization.

Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1ß (IL-1ß)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1ß maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1ß expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.

An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer.

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

Integrin-ß4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells.

Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-ß4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.

EMT, cell plasticity and metastasis.

Carcinoma cells that are induced to suppress their epithelial features and upregulate mesenchymal gene expression programs acquire traits that promote an invasive and metastatic phenotype. This is achieved through the expression of a program termed the epithelial-to-mesenchymal transition (EMT)-a fundamental cell-biological process that plays key roles in embryogenesis and wound healing. Re-activation of the EMT during cancer promotes disease progression and enhances the metastatic phenotype by bestowing upon previously benign carcinoma cell traits such as migration, invasion, resistance to anoikis, chemoresistance and tumour-initiating potential. Herein, we discuss recent insights into the function of the EMT and cancer cell plasticity during cancer progression, with a focus on their role in promoting successful completion of the later stages of the metastatic cascade.

How does multistep tumorigenesis really proceed?

Identifying the cancer cells-of-origin is of great interest, as it holds the potential to elucidate biologic mechanisms inherent in the normal cell state that have been co-opted to drive the oncogenic cell state. An emerging concept, proposed here, states that cancer stem cells, key players in cancer initiation and metastasis, arise when transit-amplifying cells with mutant genomes dedifferentiate and enter the stem cell state. This model contrasts with the notion that cancer stem cells are the direct products of neoplastically transformed normal tissue stem cells.

Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity.

The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFß. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.

Cell plasticity and heterogeneity in cancer.

BACKGROUND: Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models. CONTENT: The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity. SUMMARY: Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies.

Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-ß and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state.

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.

A perspective on cancer cell metastasis.

Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a two-phase process: The first phase involves the physical translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biological program called the epithelial-to-mesenchymal transition.

Disparate companions: tissue engineering meets cancer research.

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.