Systemic Effects of Metastatic Cancer

Parkin is an E3 ubiquitin ligase, mutated in Parkinson’s disease. Parkin is lost in many human cancers, and its overexpression inhibits tumor growth. However, the underlying basis for Parkin’s tumor suppressive role remains elusive. We observed that injecting Parkin overexpressing cells in the spleen of mice leads to reduction in metastasis. Ubiquitome analysis of Parkin overexpressing cells led to identification of two molecular targets, Mitofusin 2 (MFN2) and Transketolase (TKT). Parkin leads to ubiquitination (Ub) and degradation of MFN2 and TKT. MFN2regulates the mitochondrial machinery and alters the migratory and invasive potential of cancer cells. TKT regulates mitochondrial metabolism, thus loss of TKT by Parkin makes the cancer cells metabolically inactive. Overexpression of Parkin leads to reduction in the Pentose Phosphate Pathway (PPP) flux of cancer cells. Additionally, overexpression of Parkin leads to reduction in cell invasion and migration and a decrease in mitochondrial motility and dynamics. Parkin overexpression lead to induction of Jak-STAT1 (pSTAT1 (S727 and Y701)) and inhibition of NFKappaB (NF-kB) signaling pathways thus pointing towards an immunosuppressive role of Parkin mediated tumor suppression. Our results indicate that Parkin mediates tumor suppression by inhibiting MFN2 and TKT and by suppressing NF-kB signaling. Elucidating the roles of Parkin in immune escape and metastasis has the potential to address a significant unmet clinical need.

Aging is the major risk factor for cancer incidence, with more than 60% of cancer
diagnoses occurring in those aged 65 and above. In addition, anecdotal observations
indicate that age affects prognosis and outcome. Yet, very little experimental data dissect
the connection of aging, cancer progression and sensitivity to anti-cancer therapies. In
fact, the vast majority of preclinical research that guide the discovery of novel therapeutic
targets and clinical trials that lead to anti-cancer treatments approved by the FDA neglect
to account for the age of the average patient. This lack of understanding of how aging
plays a role in cancer is especially troubling considering that the number of new cancer
cases is on the rise globally, a fact that is tied to the increase in the proportion of the older
population in the world. Using human serum samples, we demonstrated that tumor
progression and metastasis formation occur, at least in part, as a manifestation of global
metabolic deregulation of the aged host. Our work shows that aging promotes the
increase in circulatory methylmalonic acid, a by-product of propionate metabolism, which
in turn endows cancer cells with the properties necessary to migrate, invade, survive and
thrive as metastatic lesions, emphasizing the importance of a global reprogramming of
the aged-host for tumor progression and increased cancer-associated mortality.

Metastasis is responsible for 90% of cancer-related deaths, yet the mechanisms of colonization of secondary organs remain poorly understood. For breast cancer patients, standard of care is chemotherapy, but recurrence occurs in 30% of patients. Metastasis to the liver is associated with poor prognosis and a median survival of 18-24 months. However, mechanisms driving metastatic growth in the liver after chemotherapy are understudied. Systemic chemotherapy induces hepatotoxicity and we hypothesize that chemotherapy alters the liver microenvironment, priming it as a pro-metastatic niche for colonization. To investigate effects of chemotherapy on liver ECM, we treat transgenic MMTV-PyMT mice with paclitaxel or doxorubicin.

Livers were isolated and decellularized to obtain an ECM scaffold. Metastatic breast cancer cells were reseeded on the scaffolds to mimic the arrival of metastasized cells to a post-treatment liver. Liver ECM from chemotherapy-treated mice promotes increased cancer cell migration compared to vehicle. Proteomic analysis identified a list of proteins dysregulated in chemotherapy-treated livers. Upregulated proteins such as Vitronectin and Collagen V increase cancer cell migration and invasion, features that support metastatic colonization. These data suggest that chemotherapy treatment alters the liver ECM to promote metastatic colonization. Ultimately, this study may lead to development of novel ECM- targeting drugs or biomarkers to track metastatic progression.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90- 120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non- membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. Currently, functional studies are undergoing to dissect the potential roles of the small particles in mediating systemic metabolomics reprogramming, and the medium and large particles in interacting with stromal cells and bone marrow-derived cells in pre-metastatic niches at various organ sites.

In colorectal (CRC), malignant cells are surrounded by a complex microenvironment encompassing a range of non-transformed cells, and also a diverse collection of microorganisms. Exogenous infection of animal and cellular models with specific CRC-associated bacteria, including Fusobacterium nucleatum, has supported a cancer-promoting role for members of the microbiota. We demonstrate via microbiome analysis and microbial culture that Fusobacterium species and their co-occurring microbiota, persist in liver metastasis of Fusobacterium-positive CRC. Many of the liver metastasis share the same dominant microbiome as the paired primary CRC tumors. Additionally, we have cultured Fusobacterium species from paired primary and metastatic tumors, and whole genome sequencing analysis revealed that the same strains of Fusobacterium are present in the primary tumors and distant site metastasis, despite the tissue being resected years apart. In situ hybridization analyses show that Fusobacterium is invasive in the primary tumors and metastasis, and is associated with malignant cells. We demonstrate that Fusobacterium and its co-occurring microbiota also persist and remain viable in patient derived xenografts of CRC for multiple generations in vivo. Antibiotic treatment of mice harboring these patient colon cancer xenografts led to a significant reduction in tumor Fusobacterium load, cancer cell proliferation and overall tumor growth, suggesting that microbiome modulation could change the course of this disease. Our current research centers on mapping the cellular distribution of the CRC microbiome within the tumor microenvironment and assessing the interplay between the microbiota and cancer therapeutics.

Complex regulatory mechanisms enable cell metabolism to match physiological state. The major pathways cells use to turn nutrients into energy and to synthesize macromolecules have been elucidated; however, there remain many unanswered questions regarding how metabolism supports cancer cell proliferation and thus how best to target metabolism for cancer treatment. How specific cancers use metabolism to support proliferation is determined both by cell intrinsic factors and the nutrients available within the tumor. Accumulating evidence suggests that nutrient availability in tissues is heavily influenced by non-cancer cells in the tissue such that tissue location is a major determinant of nutrient availability. Thus, to metastasize cancers have to adapt to nutrient conditions found within tissues and this can impact metastatic tumor growth. Whole body metabolism and diet can also influence nutrient availability in tissues, and comparing the effect of different diets on nutrient levels can inform the mechanism(s) for how diet alters cancer progression.

Aging and genotoxic lifestyle factors, such as smoking and obesity, disrupt organismal homeostasis, stimulating degradation of tissue microenvironments to facilitate tumorigenesis. Exercise may be prototypical strategy that maintains and restores homeostasis at the organismal, tissue, cellular and molecular levels to prevent or inhibit numerous disease conditions, including cancer. This talk will review the effects of exercise on reprogramming 'distal' tissue microenvironments (those not directly involved in the exercise response) by analyzing how alterations in the systemic milieu might modulate key tissue landscape components to influence cellular phenotypes from basic and clinical translational studies.

Brain metastases are frequent complications of breast and lung cancers and melanoma. In breast cancer, approximately 40% of metastatic patients with either HER2+ or triple negative tumors develop brain metastases. We have investigated the resistance of brain metastasis to many therapeutics, focusing on the blood-tumor barrier (BTB). We reported that the BTB is not a chaotic breakdown on the normal blood-brain barrier, but shows repeatable changes in the expression of blood-brain barrier and neuro-inflammatory response elements. These changes are seen in human craniotomy specimens and, for the sphingosine-1 phosphate 3 receptor on neuroinflammatory astrocytes, functionally modulate BTB permeability. We have studied the role of age in brain metastasis. Recent data demonstrates that brain metastases preferentially develop in younger animals as compared to older animals matched for parity. Immune profiling of metastatic brains from old and young mice revealed changes in microglial composition, and a CSF-1 receptor inhibitor significantly prevented the formation of triple negative brain metastases in young animals. Finally, our preclinical data indicates that low dose, metronomic temozolomide (TMZ) effectively prevents brain metastasis. This has lead to a Phase I/II trial of T-DM1 +/- TMZ for secondary prevention of HER2+ brain metastases.

Most breast tumors display a high degree of intratumor heterogeneity, with many distinct subpopulations of cancer cells present. Elevated diversity within a tumor increases the chance for cellular adaptation, as individual clones may react differently to changes in the tumor microenvironment. Thus, treatment of heterogeneous tumors may lead to selection of a resistant clone, its expansion and tumor progression. However, the fitness of cancer cells depends not only on their intrinsic properties, but could also be affected through interactions between different subpopulations. These interactions could be the reason for maintenance of minor clones along the major population. Therefore, intratumor heterogeneity may have functional relevance in tumor progression and colonization of metastatic sites. To emulate clonal interactions, we used the previously developed polyclonal breast cancer model of MDA-MB-468 cell line expressing soluble factors, IL-11 and FIGF.The IL-11 and FIGF clones, when present as minor population, support the growth of other clones in vivo. Moreover, polyclonal tumor with minor driving clone population arehighly metastatic. Thus, we hypothesized that clonal interactions could not only drive tumor growth, but could also play an important role in metastasis.We have found that polyclonal tumors lead to polyclonal metastases, composed of mixture of neutral and driver clones. To investigate the mechanisms of clonal interactions driving polyclonal metastasis, we performed RNA profiling of subpopulations and stroma from polyclonal tumors. Our results suggest that this cooperation is indirect and that driver clones promote metastasis by altering the tumor microenvironment. We have also found that minor driver clones affect the immune cells within primary tumor, circulating blood and metastatic site. These systemic changes significantly influence the metastatic progression. We are currently testing whether a treatment targeting this indirect clonal interaction mechanism could prevent polyclonal metastatic spread.Our study shows that the interaction between minor clones and other cancer cells could drive tumor growth and metastasis. Moreover, our results suggest that clonal cooperation in metastatic progression may be indirect and involve modulation of immune microenvironment of the primary tumor and distant organs.