Abstract
I
In this issue we depart from our usual policy of covering the full field of studies relevant to age-related disease, to instead take a more in-depth look at the topic of cancer.
Decades after the signing of the National Cancer Act and the declaration of the “War on Cancer,” major forms of the disease remain among the top killers worldwide. Although a comprehensive solution has been proposed within the SENS platform—WILT or “whole-body interdiction of lengthening of telomeres”—we have always conceded that the obstacles to implementing that strategy at the population level are fearsome indeed. Hence we remain keenly interested in the possibility that less dramatic measures might in some combination suffice to provide comparable protection.
Excluding the many artificial models involving transplants of mature neoplastic cells, cancer is now generally agreed to arise in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and a consequence of tumorigenesis; an accumulating body of evidence suggests that coevolution of the transformed cells and the surrounding stroma is an indispensable step in clinical progression. Crucially, the latter presents a far more tractable target than the cancerous population.
Using Genetically Engineered Mouse Models of Cutaneous Carcinogenesis to Characterize Oncogene-Driven Cancer Immunoediting
Bradley Kubick, PhD, University of Colorado Denver
Cancer develops as the end product of a multistep process in which cells and/or their microenvironments accumulate alterations, either genetic or epigenetic, which enable uncontrolled growth. During the early steps of this process, altered cells constituting the precursors to cancer are subject to the constraints of natural selection. As such, variations within the population of transformed cells which lead to increased relative fitness are reproductively favored by the various selective pressures of the local microenvironment. Anti-tumor immunity is an important arm of this pre-malignant niche. Consequently, for cancer to develop, at least one of the following four possibilities must occur: (1) the transformed cells progress to cancer without ever becoming immunogenic, (2) the transformed cells are immunogenic, but are able to proliferate faster than they are eliminated, (3) the host immune system becomes compromised or tolerized, or (4) from within the transformed population, new clones evolve adaptations that enable evasion of anti-tumor immunity. This final possibility has been termed “cancer immunoediting” and it includes three distinct stages of cancer-immune interaction: elimination, equilibrium, and escape. As a result of this process, every immunogenic cancer that develops in an immunocompetent host is intrinsically immune-evasive as a result of its past triumphs over anti-tumor immunity. Here, I describe a genetically engineered mouse model developed in order to study this process directly, via in vivo confocal microscopy. This model accurately recapitulates the cancer immunoediting process which occurs as a result of common mutations that drive human carcinomas. Using this model, I identified a previously unknown mechanism of early survival in which transformed cells use an immune-privileged niche as a surrogate for immune evasion. This project also provides a means to further characterize the adaptations that mediate the transitions between the stages of immunoediting. Finally, and perhaps most importantly, this model acts as a prototype for discovery of new biomarkers of human immunoediting as well as a screening tool for therapeutic interventions that could form the basis of rational cancer prevention strategies.
The role of immune surveillance in preventing cancer has long been appreciated, but a paradox remained; how can recently transformed cells, possessing only a minimal load of potentially beneficial mutations, survive long enough to develop the numerous adaptations required to resist a full-scale immune attack?
This thesis takes a major step toward remedying that ignorance, finding that such cells can exploit naturally privileged sites to hide from immune surveillance during their early development. Such sites are relatively rare; the classical examples are limited to the eye, the testes, and the placenta. The model developed here will undoubtedly prove useful in identifying markers that can be used to screen those sites for suspicious cells. Notably, at least in the case of the eye and testes, their therapeutic ablation could be relatively noninvasive.
Unfortunately for this purpose, other sites have more recently been demonstrated to possess immune privilege—most notably the articular cartilage (although there is some evidence that this is an inducible, rather than a constitutive property; indeed possibly downstream of the accumulation of senescent cells therein, a mechanism of aging that is likely to be among the first to be substantially reversed) as well as the hematopoietic stem cell niche within the bone marrow, but perhaps also including other such niches. Sadly, detecting transformed cells in these locations will be significantly less straightforward—although still not so challenging as surveying the entire body for their presence.
Molecular Pathway Involved in Breast Cancer Tumor Cell Dormancy Escape in the Bone Marrow
Louis Panzica, MS, State University of New York at Buffalo
Cancer tumor cell dormancy is a significant clinical problem in breast cancer. Many patients present detectable disseminated tumor cells at an early stage but not all of these patients will have recurrence. Also, some patients do not have detectable disseminated tumor cells but will have a recurrence years later. This poses the idea that dormancy is a major factor in cancer, but to this day there remains no methods to accurately detect and treat these cells.
In this study we use a dormancy assay to develop a list of suppressed genes that allow breast cancer cells to escape dormancy in the hematopoietic stem cell niche of the bone marrow. We use this list to rank genes based on their probable interaction with the previously confirmed dormancy gene, p38, and their role in metastasis. From known upstream or downstream genes, we were able to come across Wnt3 as a potential signaling protein inducing this dormant phenotype. Using breast cancer cells normally dormant in the hematopoietic stem cell niche (the endosteal niche), MDA-MB-231, we were able to confirm that knockdown or through natural inhibition of WNT3, causes escape from dormancy and induces proliferation. Furthermore, in breast cancer cells that normally overcome the dormant effects of the endosteal niche and actively proliferate, WNT3 overexpression returns them back to a dormant state, suppressing their proliferation.
The significance of this study is to determine signaling pathways that interact to induce a dormant state of breast cancer cells in the bone marrow. By understanding the molecular mechanisms of these cells, treatments could be developed to actively target them and prevent disease recurrence. We hypothesize that the loss of genes, such as WNT3, may be activating Myc targets, allowing for an escape from dormancy and ultimately causing a bone metastasis.
Given a fuller understanding—building on the excellent work already described—it is tempting to envisage enforcing dormancy as a means to delay the development of disease. We suspect, however, that the opposite approach will prove to be more valuable (although far more controversial). A reliable method to awaken dormant tumor cells could be employed as an adjunct to conventional therapy, potentially rendering the entire transformed population susceptible to existing chemotherapeutic drugs.
Meanwhile, the relationship between dormant tumor cells and the still-enigmatic “cancer stem cells” (CSCs) seems likely to be a fertile area of further research. Although such cells have now been identified in a range of malignancies, their proposed role as the origin of disease in vivo is less well supported. Recent findings have indicated that tumor-derived exosomes can deliver functional RNA and DNA fragments to nearby cells, whereas RNA alone can suffice to induce CSC traits, 12 raising the prospect that CSCs may arise somewhat later in the process—further victims of genetic subversion by dormant tumor cells that share their niche. Contributing further to the puzzle, evidence has now emerged that breast cancer cells can enter the dormant state after cannibalizing neighboring mesenchymal stem cells 13 —another pathway that could plausibly result in cells with both cancer-like and stem-like properties.
Inflammation Triggers Zeb1-Dependent Escape from Tumor Dormancy
Jasmine De Cock, PhD, Massachusetts Institute of Technology
Metastasis-related mortality for breast cancer patients often occurs many years after treatment of the primary tumor. Inflammation, through the orchestra of immune cells and released inflammatory cytokines, can predispose certain tissues to cancer development and can create a favorable environment for metastatic outgrowth. I evaluated whether lipopolysaccharide (LPS) could induce an inflammatory response, leading to the activation of the cell-biological epithelial-mesenchymal transition (EMT) program in dormant disseminated cancer cells in vivo, and subsequent metastatic outgrowth. To model metastatic cellular dormancy, I used a dormant subpopulation of cells (D2A1-d) that were enriched for in vivo from the highly metastatic carcinoma cell line D2A 1, that was derived from spontaneous murine mammary tumor. The ability of the EMT program to awaken dormant disseminated D2A1-d cells was directly assessed in vivo, which resulted in the formation of macro-metastases following a transient induction of either the EMT-transcription factor Snail or Zeb1. Furthermore, the transient induction of Zeb1 led to the generation of CD29+ CD24− metastasis-initiating cells. In mice bearing dormant disseminated D2A1-d cells, my findings demonstrated that LPS-treatment resulted in the awakening of D2A1-d cells and metastatic outgrowth in the lungs and bone. The awakening of dormant disseminated D2A1-d cells was dependent, albeit through unknown mechanism, on the presence of neutrophils. The LPS-mediated awakening of dormant disseminated cancer cells was also dependent upon the activation of the EMT-inducing transcription factor Zeb1 in the D2A1-d cells. In conclusion, my thesis work demonstrated that inflammation can trigger the escape of metastatic dormancy in vivo.
Yet, as we have noted on countless occasions, their remediation may be comparatively straightforward; the largest barrier is not technical, but essentially political in nature (as our elected officials remain stubbornly unconvinced that combating aging per se is in fact meritorious). Thus it is exceptionally useful, even from a purely pragmatic perspective, to gather evidence linking such “natural” derangements to the very uncontroversial target that is cancer. This thesis presents just such evidence, as well as identifying a potential target for intervention against dormancy that may be less prone to side effects than tampering with the Wnt pathway.
The requirement for neutrophils in disease progression within this system will come as no surprise to readers of this column. The discovery in 2004 that neutrophils can exocytose large fractions of their nuclear chromatin to form protease-decorated “extracellular traps” has now provided an explanation for the decades-old finding that simple DNase I injections can dramatically improve survival in animal models of metastatic disease. Although a potent immune response against external pathogens, such traps actively promote metastasis (either through the aggregation of circulating tumor cells, encouraging their expression of several resistance mechanisms, or perhaps by supplying those cells with an abundance of nucleic acids for further growth and mutation). We would thus suggest that the role of neutrophils here is not actually to awaken dormant cells, but rather is essential to their successful growth elsewhere in the body.
A Transdimensional View of Drug Resistance Evolution in Multiple Myeloma Patients
Timothy Jacobson, MSBE, University of South Florida
Multiple Myeloma (MM) is a treatable, yet incurable, malignancy of bone marrow plasma cells. This cancer affects many patients and many succumb to relapse of tumor burden despite a large number of available chemotherapeutic agents developed for therapy. This is because MM tumors are heterogeneous and receive protection from therapeutic agents by the microenvironment and other mechanisms including homologous MM-MM aggregation. Therefore, therapy failure and frequent patient relapse is due to the evolution of drug resistance, not a lack of available drugs. To analyze and understand this problem, the evolution of drug resistance has been explored and presented herein. We seek to describe the methods through which MM cells become resistant to therapy, and how this resistance evolves throughout a patient's treatment history. We achieve this in five steps.
First we review the patient's clinical history, including treatments and changes in tumor burden. Second, we trace the evolutionary tree of sub-clones within the tumor burden using standard of care fluorescence in situ hybridization (FISH). Thirdly, immunohistochemistry slides are stained and aligned to quantify the level of environmental protection received by surrounding cells and plasma in the bone marrow microenvironment (coined environment mediated drug resistance score [EMDR]). The fourth analysis type is produced through a novel 384-well plate ex vivo chemosensitivity assay to quantify sensitivity of primary MM cells to chemotherapeutic agents and extrapolate these findings to 90-day clinical response predictions. In addition to direct clinical application in the choice of best treatment, this tool was also used to study changes in sensitivity of patient tumors to other drugs, and it was observed that, upon relapse, in addition to developing resistance to the current line of therapy, tumors become cross-resistant to agents that they were never exposed to. Finally, MM-MM homologous aggregation is quantified to assess the level of drug resistance contributed by clustering of patient tumor cells, which causes upregulation of Bcl-2 expression and other resistance mechanisms.
The findings of such experimentation improve comprehension of the driving factors that contribute to drug resistance evolution on a personalized treatment basis. The aforementioned factors all contribute in varying degrees for unique patient cases, seven of which are presented in depth for this project. In summary: Environmental protection plays a critical initial role in drug resistance, which is followed by increase in tumor genetic heterogeneity as a result of mutations and drug-induced Darwinian selection. Eventually, environment-independent drug resistant subpopulations emerge, allowing the tumor to spread to unexplored areas of the bone marrow while maintaining inherited drug resistant phenotype. It is our hope that these findings will help in shifting perspective regarding optimal management of MM by finding new therapeutic procedures that address all aspects of drug resistance to minimize chance of relapse and improve quality of life for patients.
It is too soon to say with confidence whether therapies that attack cancer indirectly—whether by preventing the earliest stages of its development or by severing the essential support provided by the microenvironment—will prove significantly more effective in the clinic than the more conventional efforts of the past. Nonetheless, the current rate of progress in this direction is quite remarkable, and we do find ourselves increasingly hopeful that the “nuclear option” of WILT will yet prove unnecessary.