Cancer Dormancy as a Collective Phenomenon Across Scales in Length and Time: Biological Observations and Advanced 3D Bioengineered Models

: Patients with breast cancer can develop recurrent metastatic disease with latency periods that range from years to decades. This pause can be explained by micrometastatic disseminated tumor cell (DTC) dormancy, a stage in cancer progression in which residual disease is present but remains asymptomatic and is clinically undetectable. Cancer dormancy remains an outstanding challenge for both clinicians and scientists. Recent findings have suggested that the cancer dormancy signaling program is characterized by low ERK:p38 MAPK signaling ratio and prolonged G0/G1 arrest, reminiscent of cancer stem cells, and is thought to be a major mechanism of resistance to conventional cytotoxic chemotherapy. Cancer EMT, which is thought to precede cancer dormancy, may also contribute directly to the cancer dormancy program through its ability to induce low ERK:p38 signaling ratio. The central role of p38 in cancer dormancy was recently demonstrated by the ability of p38 inhibitors (e.g. SB203580 or dominant negatives) to break dormancy and induce tumor growth, making it an attractive therapeutic target. Here we have proposed to test a novel therapeutic concept that breast cancer dormancy can be controlled by acutely forcing dormant cancer cells into rapid proliferation, thereby rendering them sensitive to killing by cytotoxic chemotherapy. We tested the feasibility of this innovative approach by inhibiting p38 MAPK before exposing cancer cells to cytotoxic chemotherapy. We used the MTB/TAN mouse model, a Tet-inducible activated human her2/Neu model, which produces PR/ER-negative and ErbB2-independent recurrent tumors (similar to human triple negative breast cancer) after withdrawal of doxycycline (deinduction). These recurrent tumors have all the characteristic of having undergone EMT and are relatively growth arrested and dormant.

Patients with cancer can develop recurrent metastatic disease with latency periods that range from years even to decades. This pause can be explained by cancer dormancy, a stage in cancer progression in which residual disease is present but remains asymptomatic. Cancer dormancy is poorly understood, resulting in major shortcomings in our understanding of the full complexity of the disease. Here, I review experimental and clinical evidence that supports the existence of various mechanisms of cancer dormancy including angiogenic dormancy, cellular dormancy (G0-G1 arrest) and immunosurveillance. The advances in this field provide an emerging picture of how cancer dormancy can ensue and how it could be therapeutically targeted.

<div>Abstract<p>Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors.</p>Implications: <p>We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.</p></div>

<div>Abstract<p>Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors.</p>Implications: <p>We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.</p></div>

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors.Implications: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.

Cellular stress responses and metabolic reprogramming in cancer progression and dormancy.

In this paper a mathematical model describing the growth of a solid tumour in the presence of an immune system response is presented. In particular, attention is focused upon the attack of tumour cells by so-called tumour-infiltrating cytotoxic lymphocytes (TICLs), in a small, multicellular tumour, without necrosis and at some stage prior to (tumour-induced) angiogenesis. At this stage the immune cells and the tumour cells are considered to be in a state of dynamic equilibrium--cancer dormancy--a phenomenon which has been observed in primary tumours, micrometastases and residual disease after ablation of the primary tumour. Nonetheless, the precise biochemical and cellular mechanisms by which TICLs control cancer dormancy are still poorly understood from a biological and immunological point of view. Therefore we focus on the analysis of the spatio-temporal dynamics of tumour cells, immune cells and chemokines in an immunogenic tumour. The lymphocytes are assumed to migrate into the growing solid tumour and interact with the tumour cells in such a way that lymphocyte-tumour cell complexes are formed. These complexes result in either the death of the tumour cells (the normal situation) or the inactivation (sometimes even the death) of the lymphocytes. The migration of the TICLs is determined by a combination of random motility and chemotaxis in response to the presence of chemokines. The resulting system of four nonlinear partial differential equations (TICLs, tumour cells, complexes and chemokines) is analysed and numerical simulations are presented. We consider two different tumour geometries--multi-layered cell growth and multi-cellular spheroid growth. The numerical simulations demonstrate the existence of cell distributions that are quasi-stationary in time and heterogeneous in space. A linear stability analysis of the underlying (spatially homogeneous) ordinary differential equation (ODE) kinetics coupled with a numerical investigation of the ODE system reveals the existence of a stable limit cycle. This is verified further when a subsequent bifurcation analysis is undertaken using a numerical continuation package. These results then explain the complex heterogeneous spatio-temporal dynamics observed in the partial differential equation (PDE) system. Our approach may lead to a deeper understanding of the phenomenon of cancer dormancy and may be helpful in the future development of more effective anti-cancer vaccines.

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. The identification of genes and proteins that are specifically involved in the collective behavior of cancer cells has thus far been limited. Here, we studied the role of the actin-binding protein Girdin in collective cancer cell migration. This protein is a specific regulator of collective migration of neuroblasts born in the subventricular zone of the postnatal and adult brains and participates in the progression of cancer. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to β-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the β-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Citation Format: Xiaoze Wang, Atsushi Enomoto, Liang Weng, Hisashi Haga, Sumire Ishida, Masahide Takahashi. The actin-binding protein Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3160.

The hiatus observed in the progression of cancer after diagnosis and treatment in a large proportion of patients has led to the notion that a state of cancer dormancy must exist during tumor progression. However, research on this stage of cancer has been limited due to the lack of appropriate models and clinical correlates. Fortunately, the last decade has seen the development of new cancer dormancy models, whole animal and intravital imaging techniques and the molecular characterization of minimal residual disease. These studies enabled researchers to reveal intriguing mechanisms and molecular determinants that define tumor dormancy. It is imperative to understand the basic mechanisms of dormancy, as this will accelerate the development of new markers of progression and novel therapeutic opportunities to induce dormancy and/or eradicate dormant disease. This issue of Cell Cycle includes a “Spotlight on Cancer Dormancy” highlighting major contributions to the field of cancer dormancy from basic and clinical studies. We anticipate that this will initiate a forum of discussion on the problem of cancer dormancy and stimulate investigators to study this rather unexplored but undeniably relevant clinical stage of cancer progression.

Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin to form the autophagy initiating complex, inducing ATG4 and decorating the autophagosome membrane, co-localizing with LC3II. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Outgrowth can be delayed if dormant cancers are treated with chloroquine, a functional inhibitor of autophagy, suggesting that autophagy is a survival mechanism in this context. Our current experiments were designed to determine whether autophagic cells could be eliminated more or less readily by treatment with cisplatin with or without chloroquine. In short-term or long-term cell culture, induction of ARHI enhanced the cytotoxic effect of cisplatin and tumor cell killing was further enhanced by the addition of chloroquine. In xenografts, treatment with cisplatin slowed the outgrowth of dormant autophagic ovarian cancer cells, but the addition of chloroquine did not further inhibit xenograft growth. In cell culture, caspase 3/8 release and Anexin V expression on the surface of ovarian cancer cells can account for enhancement of caspase dependent apoptosis observed when autophagic ovarian cancer cells are treated with cisplatin. Treatment of ARHI-induced autophagic ovarian cancer cells with chloroquine and cisplatin downregulated the expression of survivin, XIAP and Bcl-2. Re-expression of ARHI induced caspase dependent apoptosis and autophagic cell death in the absence of cisplatin. ARHI-induced apoptosis and autophagic cell death could be blocked by Z-VAD that inhibits caspase mediated apoptosis and by stable knockdown of ATG5 that inhibits autophagy. In this context, induction of autophagy enhanced platinum toxicity. Additional treatment with chlorooquine produced additional cancer cell cytotoxicity in cell culture, but not in dormant xenografts. These preclinical studies do not support the addition of chloroquine to platinum based chemotherapy for ovarian cancer in the clinic, but do support the association of “autophagic” cell death with apoptosis. Citation Format: Michele N. Washington, Grace Suh, Aaron F. Orozco, Maojie Yang, Yan Wang, Neely Atkinson, Warren Liao, Zhen Lu, Robert Bast. ARHI-induced autophagy enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1680. doi:10.1158/1538-7445.AM2013-1680

We demonstrate the first application of Raman spectroscopy in diagnosing nonmalignant, premalignant, malignant, and metastatic stages of breast cancer in a three-dimensional (3-D) cell culture model that closely mimics an in vivo environment. Comprehensive study comparing classification in two-dimensional (2-D) and 3-D cell models was performed using statistical methods composed of principal component analysis for exploratory analysis and outlier removal, partial least squares discriminant analysis, and elastic net regularized regression for classification. Our results show that Raman spectroscopy with an appropriate classification tool has excellent resolution to discriminate the four stages of breast cancer progression, with a near 100% accuracy for both 2-D and 3-D cell models. The diversity in chemical groups related to nucleic acids, proteins, and lipids, among other chemicals, were identified by appropriate peaks in the Raman spectra that correspond to the correct classification of the different stages of tumorigenesis model comprising of MCF10A, MCF10AneoT, MCF10CA1h, and MCF10CA1a cell lines. An explicit relationship between wavenumber and the stages of cancer progression was identified by the elastic net variable selection.

BACKGROUND Lung cancer is the most common primary tumor to metastasize to the brain. Poor understanding of the brain metastasis process limits therapeutic options. Despite emerging data on the role of YAP signaling pathway in the development of brain tumors, a gap in knowledge remains regarding its role in brain colonization. We hypothesize that YAP1 and the associated TEAD transcriptional factors are responsible for lung cancer progression and that intervention with YAP/TEAD-targeted therapy will prevent brain dissemination. METHODS Lung cancer cells at different stages of cancer progression (VMRC-LCD and NCI-H1975, lung cancer cell lines; NCI-H1915 and LN001, brain metastatic cancer cells) were genetically engineered to overexpress or silence/knockout YAP1 using lentiviral transduction of constructs expressing shRNA/sgRNA or YAP1. We tested several tumor cell capabilities using well-established in vitro assays, including invasion and migration assays, and an in vivo brain metastasis (BM) model to determine the biological effects of YAP1/TEAD suppression at different stages of lung cancer progression. Dose-response assays were performed to determine IC50 of the TEAD inhibitor, verteporfin. RESULTS YAP overexpression enhances the proliferative phenotype in metastatic and BM cell lines but not in non-metastatic cells. However, YAP overexpression only increases the migration ability of BM cell lines such as LN001 and NCI-H1915 compared to the metastatic NCI-H1975 and non-metastatic lung cancer cells. Moreover, we showed the efficacy of the YAP1/TEAD inhibitor in patient-derived lung BM cells. Finally, our in vivo preliminary data showed YAP1 involvement in lung cancer cell colonization of the brain but not in sustaining brain metastasis growth. CONCLUSIONS Our findings showed YAP1 involvement in lung cancer cell colonization of the brain, suggesting YAP1 inhibition is a clinically actionable target for preventing lung cancer dissemination and providing a strong rationale to further evaluate the benefit of a YAP1/TEAD-targeted therapy in in vivo models of BM.

Recurrent disease is the most daunting aspect of cancer treatment; however, how tumor cells become dormant and later recur even years after “successful” treatment is poorly understood. However, studies to decipher mechanisms responsible for dormancy have been hampered due to lack of appropriate model. In this study, we isolated two syngeneic cell lines (Indolent and Aggressive), which recapitulates dormancy and recurrence of prostate cancer in the bone microenvironment. We found that Indolent cells retained dormant phenotype whereas Aggressive cells grew rapidly in the tibial bone in vivo whereas the in vitro cell proliferation, invasion, migration and self-renewal properties of both cells in culture were not altered, suggesting the role of microenvironment in regulation of dormancy and recurrence. The expression profile by our microarray analysis revealed that SPARC and Noggin (a known inhibitor of BMP7) were significantly upregulated in Indolent and Aggressive cells, respectively. SPARC secreted by Indolent cells was found to stimulate BMP7 expression in bone stromal cells that in turn inhibited cancer cells by activating the dormancy-associated p38 MAPK pathway and its downstream cell cycle inhibitors, p21 and p18. In addition, BMP7 increased senescence and diminished stem-cell phenotype of cancer cells, which was rescued by addition of recombinant Noggin. We also found that BMPR2 plays a crucial role in SPARC-induced paracrine inhibition of tumor cells residing in the bone. Accordingly, BMPR2 knockdown rescued the BMP7-mediated decrease in stemness. Importantly, the BMPR2 correlative signature was enriched in patients who did not experience recurrence for a long period of time, which further verified the role of BMPR2 downstream signaling in dormancy. When primary tumor samples were examined by immunohistochemistry, both SPARC and BMPR2 were found to be significantly downregulated in patients with bone metastasis. Moreover, patients who did not experience bone metastasis were found to express high level of both SPARC and BMPR2. Importantly, we also observed elevated expression of DNA methylase genes, DNMT1 and -3B in Aggressive cells. Treatment of Aggressive cells with NS398, a COX-2 inhibitor downregulated DNMTs and concomitantly augmented SPARC expression in vitro. Therefore, recurrence of cancer cells in the bone microenvironment involves epigenetic regulation of SPARC and disruption of inhibitory crosstalk of cancer cells with the stroma. These findings suggest that SPARC plays a critical role in maintaining dormancy of prostate cancer cells in bone microenvironment. Citation Format: Sambad Sharma, Fei Xing, Kerui Wu, Yin Liu, Kounosuke Watabe. SPARC expression stimulates paracrine inhibitory response from bone marrow stroma during dormancy of prostate cancer in the bone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4098.

Multiple roles of p53 in cancer development: Regulation of tumor microenvironment, m6A modification and diverse cell death mechanisms

Metastasis, the process by which cancers progress to spread to surrounding tissues and distant organs, is the primary cause of death in most cancers. Characterizing the regulatory mechanisms underlying metastasis is of great importance for identifying novel therapeutic targets. Reconstructing gene regulatory networks (GRNs) from high throughput multi-omics data obtained from various stages of cancer progression has great potential for aiding this challenging undertaking. In this study, we developed a computational framework to characterize the major regulatory mechanisms underlying colorectal cancer (CRC) progression by simultaneous analysis of dynamic gene expression and histone modification profiles that have been collected from SW480 cell lines selected for various levels of invasiveness, along with transcription factor (TF) binding (ChIP-seq) data from the HCT116 CRC cell line available from the ENCODE project. We built upon our previously developed probabilistic graphical model for integration of multi-omic data, called pGENMi, to identify TFs involved in CRC metastasis. Our framework considers differential expression of each gene along with evidence of that gene being under the regulatory influence of a specific TF. Data are aggregated across all genes to assign a probabilistic significance to each TF. Importantly, the cis -regulatory evidence is based on progression-associated changes in histone marks within a TF’s ChIP peak. The model simultaneously includes all TFs for which binding data are available, permitting the assessment of significance while accounting for the influence of other TFs. Disruption of JUND, one of the highest ranked TFs in our analyses, confirmed involvement in the CRC invasive phenotype. The importance of considering histone modification status for these analyses is underscored by the inability to identify JUND as a significant TF when dynamic epigenomic data were ignored. In addition to finding the TFs associated with cancer progression, our framework identified candidate genes predicted to mediate the effects of TFs on cancer progression, thus enabling the reconstruction of the underlying GRN. We used a gene set derived from this predicted GRN as a signature to subtype CRC patient profiles from TCGA and found the subtypes to have significantly different survival outcomes.