Contribution of the Microenvironment in Bone Resident Cancer: the Role of Thrombospondin-1 in Bone and Risk Loci Contributing to Multiple Myeloma

Cancer growth in bone, a defining characteristic of multiple myeloma and common in many solid tumors including breast and prostate cancer, is characterized by the active participation of host microenvironment cells. Tumor cells stimulate the vicious cycle through stimulation of bone remodeling by osteoclasts and osteoblasts, resulting in increased release of growth factors and cytokines to further promote local tumor growth. Notably, cancer cells are unable to remodel bone independently, and require the metabolically active bone cells for this support. A specific role for the bone microenvironment in mediating tumor growth was first discussed in 1889 with the introduction of Paget\u27s seed-and-soil hypothesis that tumor cells will only grow in a favorable microenvironment. This dissertation is focused on investigating the properties of a congenial soil. In Chapter 2 we discuss the role of bone-resident Thrombospondin-1 (TSP1), a glycoprotein with well-documented roles in promoting cancer growth. Chapter 3 is focused on the identification of genes conferring inherited susceptibility to multiple myeloma that participate both in malignant cells and in host supportive cells. TSP1 is a large matricellular protein that interacts β3 integrin, CD47, and other receptors to regulate cell migration, adhesion, and proliferation with effects on tumor angiogenesis, inflammation, and wound healing. We examined the role of TSP1 in the bone microenvironment. TSP1-/- mice had increased trabecular bone volume and increased cortical bone area and thickness compared to wild type controls. Surprisingly, TSP1-/- mouse bones did not resist bending as much as anticipated, and we confirmed a bone materials defect, indicating that TSP1 was important for maintaining bone quality. In addition to the mechanical defects, TSP1-/- mice had decreased serum CTX compared to controls, indicating an osteoclast defect. Primary osteoclast cultures require TSP1 at early stages of osteoclastogenesis, though exogenous TSP1 is dispensable at later stages of maturation. Interestingly, we found that TSP1-deplete osteoclast cultures had a significant and dramatic increase in inducible nitric oxide synthase (iNOS) expression. Moreover, upon administration of a NOS inhibitor to mice, the TSP1-/- bone resorption defect was rescued to wild type levels. To test whether osteoblast-deposited bone-resident TSP1 inhibits early osteoclast NO signaling, we plated osteoclasts on either wild type or TSP1-/- bone and found that only wild type bone was sufficient to block iNOS expression. Thus, we conclude that TSP1 is a paracrine signaling molecule that couples OB activity to OC formation and targeting of the TSP1 signaling pathway, currently under investigation for treating cancer and modulating blood pressure, may have additional effects on bone. The second part of this dissertation focuses on host susceptibility to multiple myeloma (MM) and its requisite precursor, monoclonal gammopathy of undetermined significance (MGUS). The C57Bl/KaLwRij (KaLwRij) mouse strain has increased susceptibility to benign idiopathic paraproteinemia (BIP), analogous to human MGUS, and is a common mouse model for the disease. Following immunization, KaLwRij mice developed a sustained M-spike at a higher frequency than 12 other genetically diverse mouse strains, most notably, the highly related C57Bl/6J (B6) strain. To query the genetic basis for BIP susceptibility in KaLwRij mice, we completed SNP analysis to identify variation between KaLwRij and B6. We found that KaLwRij is a unique inbred strain, completely distinct from the closely related B6. We identified several thousand variants between KaLwRij and B6 and compiled a candidate gene list of 419 genes. To ensure that we would pursue genes applicable to human disease, we completed whole genome association analysis between MM patients and healthy controls and identified SNPs located in 180 gene loci. Combining these two gene sets resulted in a candidate gene list of 5 genes, one of which, Samsn1, is annotated as a negative regulator of B-cell activation. Upon closer examination of the Samsn1 locus, we found that KaLwRij harbored a germline deletion of all coding exons of the gene, resulting in absent RNA and protein expression. Consistent with the published role of SAMSN1 in genetically deficient mice, KaLwRij mice had enhanced B-cell function as evaluated by B-cell proliferation in primary culture and in vivo IgG2b response. To investigate the role of Samsn1 in host cells, we profiled Samsn1 expression in bone microenvironment cells and found that macrophages were high expressers. KaLwRij macrophages had enhanced expression of polarization markers, suggesting that they are pre-activated under homeostatic conditions. Further, we found that KaLwRij macrophages stimulated to a pro-tumorigenic M2 polarization promoted 5TG myeloma tumor growth more than B6 control macrophages in mice. In conclusion, we identified candidate gene lists underlying murine and human susceptibility to MGUS and myeloma. Specifically, we found that Samsn1 contributes to KaLwRij susceptibility to BIP, and likely to human MGUS, through effects in pre-malignant B-cells and in macrophages

Poly‐aneuploid cancer cells promote evolvability, generating lethal cancer

Cancer cells utilize the forces of natural selection to evolve evolvability allowing a constant supply of heritable variation that permits a cancer species to evolutionary track changing hazards and opportunities. Over time, the dynamic tumor ecosystem is exposed to extreme, catastrophic changes in the conditions of the tumor—natural (e.g., loss of blood supply) or imposed (therapeutic). While the nature of these catastrophes may be varied or unique, their common property may be to doom the current cancer phenotype unless it evolves rapidly. Poly‐aneuploid cancer cells (PACCs) may serve as efficient sources of heritable variation that allows cancer cells to evolve rapidly, speciate, evolutionarily track their environment, and most critically for patient outcome and survival, permit evolutionary rescue, therapy resistance, and metastasis. As a conditional evolutionary strategy, they permit the cancer cells to accelerate evolution under stress and slow down the generation of heritable variation when conditions are more favorable or when the cancer cells are closer to an evolutionary optimum. We hypothesize that they play a critical and outsized role in lethality by their increased capacity for invasion and motility, for enduring novel and stressful environments, and for generating heritable variation that can be dispensed to their 2N+ aneuploid progeny that make up the bulk of cancer cells within a tumor, providing population rescue in response to therapeutic stress. Targeting PACCs is essential to cancer therapy and patient cure—without the eradication of the resilient PACCs, cancer will recur in treated patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156440/2/eva12929_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156440/1/eva12929.pd

Phytobiomes are compositionally nested from the ground up

Plant-associated microbes are critical players in host health, fitness and productivity. Despite microbes’ importance in plants, seeds are mostly sterile, and most plant microbes are recruited from an environmental pool. Surprisingly little is known about the processes that govern how environmental microbes assemble on plants in nature. In this study we examine how bacteria are distributed across plant parts, and how these distributions interact with spatial gradients. We sequenced amplicons of bacteria from the surfaces of six plant parts and adjacent soil of Scaevola taccada, a common beach shrub, along a 60 km transect spanning O’ahu island’s windward coast, as well as within a single intensively-sampled site. Bacteria are more strongly partitioned by plant part as compared with location. Within S. taccada plants, microbial communities are highly nested: soil and rhizosphere communities contain much of the diversity found elsewhere, whereas reproductive parts fall at the bottom of the nestedness hierarchy. Nestedness patterns suggest either that microbes follow a source/sink gradient from the ground up, or else that assembly processes correlate with other traits, such as tissue persistence, that are vertically stratified. Our work shines light on the origins and determinants of plant-associated microbes across plant and landscape scales

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co???separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density???gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co???separate contaminants when the non???EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non???EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios

Drug-resilient cancer cell phenotype is acquired via polyploidization associated with early stress response coupled to HIF-2α transcriptional regulation

Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF-2α. We found altered chromatin accessibility, ablated expression of RB1, and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF-2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF-2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF-2α in stress-response by polyploidy suggest a novel avenue for tackling chemotherapy-induced resistance in cancer

Antagonizing Integrin β3 Increases Immunosuppression in Cancer

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8+ T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8+ T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484–95. ©2016 AACR

Epidemiology of basal-like breast cancer

Risk factors for the newly identified “intrinsic” breast cancer subtypes (luminal A, luminal B, basal-like and human epidermal growth factor receptor 2-positive/estrogen receptor-negative) were determined in the Carolina Breast Cancer Study, a population-based, case–control study of African-American and white women. Immunohistochemical markers were used to subtype 1,424 cases of invasive and in situ breast cancer, and case subtypes were compared to 2,022 controls. Luminal A, the most common subtype, exhibited risk factors typically reported for breast cancer in previous studies, including inverse associations for increased parity and younger age at first full-term pregnancy. Basal-like cases exhibited several associations that were opposite to those observed for luminal A, including increased risk for parity and younger age at first term full-term pregnancy. Longer duration breastfeeding, increasing number of children breastfed, and increasing number of months breastfeeding per child were each associated with reduced risk of basal-like breast cancer, but not luminal A. Women with multiple live births who did not breastfeed and women who used medications to suppress lactation were at increased risk of basal-like, but not luminal A, breast cancer. Elevated waist-hip ratio was associated with increased risk of luminal A in postmenopausal women, and increased risk of basal-like breast cancer in pre- and postmenopausal women. The prevalence of basal-like breast cancer was highest among premenopausal African-American women, who also showed the highest prevalence of basal-like risk factors. Among younger African-American women, we estimate that up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity

The issues with tissues : the wide range of cell fate separation enables the evolution of multicellularity and cancer

Our understanding of the rises of animal and cancer multicellularity face the same conceptual hurdles: what makes the clade originate and what makes it diversify. Between the events of origination and diversification lies complex tissue organization that gave rise to novel functionality for organisms and, unfortunately, for malignant transformation in cells. Tissue specialization with distinctly separated cell fates allowed novel functionality at organism level, such as for vertebrate animals, but also involved trade-offs at the cellular level that are potentially disruptive. These trade-offs are under-appreciated and here we discuss how the wide separation of cell phenotypes may contribute to cancer evolution by (a) how factors can reverse differentiated cells into a window of phenotypic plasticity, (b) the reversal to phenotypic plasticity coupled with asexual reproduction occurs in a way that the host cannot adapt, and (c) the power of the transformation factor correlates to the power needed to reverse tissue specialization. The role of reversed cell fate separation for cancer evolution is strengthened by how some tissues and organisms maintain high cell proliferation and plasticity without developing tumours at a corresponding rate. This demonstrates a potential proliferation paradox that requires further explanation. These insights from the cancer field, which observes tissue evolution in real time and closer than any other field, allow inferences to be made on evolutionary events in animal history. If a sweet spot of phenotypic and reproductive versatility is key to transformation, factors stimulating cell fate separation may have promoted also animal diversification on Earth