Browsing by Subject "Metastasis"

Ovarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of ovarian cancer related deaths. Despite the clinical significance of OC omental metastases, the precise molecular mechanisms which drive this phenomenon have not been well characterized, making the resulting aggressive phenotype even more puzzling. Recent evidence has highlighted the importance of metabolic reprograming in driving this tumoral behavior, with OC metastases adapting to utilize nutrients available in the metastatic niche to rapidly proliferate. To better understand the metabolic changes that underlie the aggressive nature of OC, we undertook a broad investigation to better characterize metabolic reprogramming in ovarian cancer, with a focus on omental metastasis and chemoresistance. Firstly, we sought to expand the arsenal of tools used to study OC metabolism. In particular, we were interested in using organoids, self-organizing, expanding 3D cultures derived from stem cells, to study OC. Using tissue derived from patients, these miniaturized models have been shown to recapitulate various aspects of patient physiology and disease phenotypes including genetic profiles and drug sensitivities. However, as metabolism modeling in these 3D cultures remains yet unexplored, we aimed to introduce genetically encoded, fluorescent biosensors as robust tools to interrogate metabolism in this context. In Chapter 2, we detail our investigation in which we transfected plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n=8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC. Secondly, in Chapter 3, we focused on characterizing the role of the pentose phosphate pathway (PPP), a metabolic pathway responsible for producing nucleotide pentose precursors through a nonoxidative series of reactions and the reducing equivalent NADPH through a distinct oxidative branch. Using computational analysis of gene expression data, metabolomics analysis, and biochemical approaches, we observed upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, of metastatic OC cells in the omentum compared to primary OC tumors. We established these increases coincided with increased oxidative stress experienced by OC cells in the omental microenvironment, using both established oxidative stress assays and genetically encoded biosensors; and sought to understand if the PPP was an important cellular mechanism to compensate for this metabolic pressure. Indeed, both shRNA-mediated and pharmacological inhibition of G6PD, the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting this adaptive metabolic dependency is important for OC omental metastasis. This work collectively illustrates the importance of characterizing OC metabolism and supports future efforts to develop tools to more effectively investigate and target aspects of metabolic reprogramming in OC which drive metastasis and chemoresistance.

The ABL family of non-receptor tyrosine kinases, ABL1 (also known as c-ABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration and invasion. ABL tyrosine kinases play an oncogenic role in human leukemias. However, the role of ABL kinases in solid tumors including breast cancer progression and metastasis is just emerging.

To evaluate whether ABL family kinases are involved in breast cancer development and metastasis, we first analyzed genomic data from large-scale screen of breast cancer patients. We found that ABL kinases are up-regulated in invasive breast cancer patients and high expression of ABL kinases correlates with poor prognosis and early metastasis. Using xenograft mouse models combined with genetic and pharmacological approaches, we demonstrated that ABL kinases are required for regulating breast cancer progression and metastasis to the bone. Using next generation sequencing and bioinformatics analysis, we uncovered a critical role for ABL kinases in promoting multiple oncogenic pathways including TAZ and STAT5 signaling networks and the epithelial to mesenchymal transition (EMT). These findings revealed a role for ABL kinases in regulating breast cancer tumorigenesis and bone metastasis and provide a rationale for targeting breast tumors with ABL-specific inhibitors.

Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-β) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-β components are common across a variety of cancers. In particular, the expression of the type III TGF-β receptor (TβRIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-β on carcinogenesis, this dissertation looks at the role of TβRIII in breast cancer progression.

Through an examination of clinical specimens, loss of TβRIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low TβRIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for TβRIII expression. The contribution of TβRIII to tumor progression was further examined by examining known TGF-β functions, including proliferation, apoptosis, migration, and invasion. TβRIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis in vivo, as mammary cancer cells stably expressing TβRIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. In vitro, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble TβRIII (sTβRIII) was shown to recapitulate the suppressive effects on invasion.

To further explore other potential mechanisms by which TβRIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of TβRIII, which is known to be critical in the regulation of TβRIII cell surface expression and downstream signaling. In vitro, I demonstrated that abrogation of the cytoplasmic domain attenuates the TβRIII-mediated suppression of migration and invasion. TβRIII's suppressive effects are also concomitant with loss of TGF-β signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-β signaling while the full length receptor was able to do so. In vivo, I also showed that in the absence of the cytoplasmic domain, TβRIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of TβRIII with the scaffolding protein GIPC also attenuated TβRIII's ability to dampen TGF-β signaling and invasion.

In sum, TβRIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to TβRIII's tumor suppressive effects, namely the generation of sTβRIII and the interaction of TβRIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-β signaling in cancer biology, elucidate mechanisms by which TβRIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of TβRIII in regulating tumor biology in general.

Basement membranes are dense, sheet-like forms of extracellular matrix that

surround the epithelial tissues of metazoan organisms. While these structures are

critical for epithelial support and tissue organization, basement membranes also pose

formidable barriers to most cells. However, certain specialized cells are able to breach

these barriers and move between tissues. Acquisition of cell invasive behavior by some

tumor cells is thought be an important step in cancer progression. Due to the clear basic

and clinical importance of understanding the mechanisms underlying cell invasion

through basement membranes, cell invasive behaviors has been an area of intense study.

In this work I examine a developmentally regulated model of cell invasive behavior in

the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels

basement membrane to connect two epithelial tissues, the uterus and vulva. Using this

model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.

Cancer cell metastasis to the leptomeninges (LM) is a rapidly fatal disease complication. Despite recent advances in treating brain parenchymal metastasis, standard of care for leptomeningeal disease (LMD) has remained essentially unchanged for decades. The paucity of targeted molecular therapies to treat LMD is attributed to our poor understanding of the molecular mechanisms governing LM invasion. We previously showed that acute lymphoblastic leukemia (ALL) cells can invade the LM by abluminal migration along emissary vessels that connect vertebral/calvarial bone marrow to meninges. This process is dependent on ALL cell integrin α6 engagement with vascular basement membrane laminin. To study whether bone metastatic breast cancer could invade the LM along this pathway, we developed a new syngeneic mouse model of breast cancer LMD and also utilized the xenograft 1833 breast cancer model in which intracardiac engrafted mice develop bone metastasis and LMD. We used these models in combination with cutting-edge techniques including intravital microscopy (IVM) of the LM and high-resolution microCT to study LMD at a single-cell level. Integrin α6 deletion in breast cancer cells (BCCs) resulted in prolonged survival and a decrease in LMD burden in both a xenograft and syngeneic mouse model. Histologic analysis, tissue immunofluorescence, and microCT of tumored mice revealed BCCs in transit along the abluminal surface of emissary vessels connecting the bone marrow and LM. IVM of LM also revealed that >95% of BCCs co-localized with CSFR1+ meningeal macrophages. We found evidence of crosstalk between BCCs and LM macrophages, as breast cancer invasion led to a two-fold upregulation of GDNF, a key neurotrophic factor released by macrophages in response to neuronal injury. In support of an important functional role for BCC-macrophage interactions in tumor cell survival, specific ablation of this CSFR1+ population in vivo markedly reduced immune cell derived-GDNF and resulted in prolonged LMD-free survival. In our previously published models of ALL we found that emissary vessel trafficking is dependent on PI3Kδ regulation of integrin α6 expression and activation of cellular migration pathways; however, ALL cells did not rely on PI3Kδ signaling for growth. We therefore explored the effect of targeting multiple PI3K isoforms with the pan-PI3K inhibitor, copanlisib. Using multiple mouse models of ALL we found that copanlisib treatment induced a growth arrest in ALL cells thus reducing systemic disease burden in mice. Moreover, we found that copanlisib sensitized ALL cells to chemotherapy and reduced systemic and CNS disease burden by inhibiting PI3K/Akt-dependent survival pathways activated upon cellular stress. Taken together, our data suggest that BCCs co-opt neuronal pathfinding mechanisms and resident macrophages to efficiently invade and thrive within the LM niche. Our data also reveal the promising, multifaceted potential of pan-PI3Ki for ALL CNS prophylaxis, systemic disease control, and chemosensitization.

Cancer metastasis is the cause of about 90% of cancer patients' deaths. Despite significant improvements in the past three decades in understanding the molecular bases of oncogenic transformation of cancer cells, little is known about the molecular mechanisms underlying tumour cells' alteration of their microenvironment, entrance into the circulation, and colonization of distant organs. In recent years, accumulating evidence has indicated that tumour microenvironment, which consists of a variety of cell types and extracellular matrix components，plays an important role in regulating the metastatic abilities of carcinoma cells. Co-opted by cancer cells, those stromal cells promote tumour progression via multiple mechanisms, including enhancement of tumour invasiveness, elevation of angiogenesis, and suppression of immune surveillance activity.

Using a series of human breast cancer cell lines with different metastatic potentials in vivo, we performed an unbiased screen examining expression of miRNAs, and found that miR-126 and miR-126*, whose expression are regulated by methylation of the promoter of their host gene Egfl7 inside tumour cells, were significantly negatively correlated with metastatic potential. Using both mouse xenograft models and in vitro assays, we showed that this pair of miRNAs suppressed breast cancer metastasis through shaping the tumour microenvironment without changing tumour cell autonomous properties. Specifically, miR-126 and miR-126* act independently to suppress the sequential recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes into the primary tumour stroma, consequently inhibiting lung metastasis by breast tumour cells. Mechanistically, these miRNAs directly inhibit the production of stromal cell-derived factor-1 alpha (Sdf-1α, also known as Cxcl12), and indirectly suppress the expression of chemokine (C-C motif) ligand 2 (Ccl2) by the cancer cells within the tumour mass in an Sdf-1α-dependent manner. In addition, in contrast with the majority of reports which have shown incorporation of only the guiding strand of the miRNA duplex into the mRNA-targeting RNA induced silencing complex (RISC), both strands of the miR-126 RNA duplex are maintained at a similar level and suppress Sdf-1α expression independently.

Collectively, we have determined a dynamic process by which the composition of the primary tumour microenvironment could be altered via a change in the expression of two tumour-suppressive miRNAs derived from a single miRNA precursor to favor metastasis by breast cancer cells. Importantly, this work provides a prominent mechanism to explain the clinical correlation between reduced expression of miR-126/126* and poor metastasis-free survival of breast cancer patients.

Signaling from the G protein, Rap1 is involved in several fundamental biological processes. Yet the mechanism or even consequence of Rap1 signaling in several biologies and diseases is still unclear. Rap1 has been implicated in cancer tumorigenesis, but its role in cancer invasion and metastasis is less understood. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting that Rap1 may promote several processes associated with metastasis. Recent studies in another biological system have demonstrated that the Rap activator proteins, Epac, are important regulators of pancreatic β-cell insulin secretion. However, the role of Rap1 in β-cell biology has not yet been defined. Here we established roles for Rap1 in distinct signaling events and begin to answer some of the key questions about Rap1 function in two diverse biologies: cancer metastasis and pancreatic islet β-cell function.

Elucidating the mechanisms of prostate and breast cancer survival and metastasis are critical to the discovery of novel therapeutic targets. Examination of prostate cancer cell lines revealed cells with a high metastatic ability exhibited increased Rap1 activity and reduced expression of the negative regulator, Rap1GAP. Activation of Rap1 increased prostate and breast cancer cell migration and invasion, and inhibition of Rap1A activity via RNAi-mediated knockdown or ectopic expression of Rap1GAP markedly impaired cancer cell migration and invasion. Additional studies implicated integrins α4, β3, and αvβ3 in the mechanism of Rap1-mediated prostate and breast cancer migration. Furthermore, these same integrins and matrix metalloproteinases were shown to be involved in Rap1-induced prostate cancer invasion. Introduction of activated Rap1 into prostate cancer cells dramatically enhanced the rate and incidence of CaP metastasis in a mouse metastasis model. In another mouse xenograft model, blockade of Rap1 signaling by expression of Rap1GAP abrogated breast cancer metastastasis. These studies support a role for aberrant Rap1 activation in prostate and breast cancer metastatic progression, and suggest that targeting Rap1 signaling could provide a means to control metastatisis of these cancers.

In a seperate biological system, the effects of Rap1 signaling on pancreatic β-cells was directly examined. Activation of Rap1 was demonstrated to promote ribosomal protein S6 phosphorylation through the mTOR and p70 S6 kinase (S6K1) pathway, a known growth-regulatory pathway. This newly defined β-cell axis acts downstream of cAMP, in parallel with the stimulation of both Epac and PKA. Like previous studies on Epac, activation of Rap1 indeed increased glucose stimulated insulin secretion (GSIS) from rat islet β-cells; however, Rap1-mediated GSIS did not appear to signal through this new S6 pathway. Interestingly, Rap1 was show to significantly increase islet cell proliferation and this indeed occured through signaling to mTOR and S6. In summary, these findings represent a new link between cAMP signaling and the pathways controlling β-cell proliferation, and suggest that directly targeting this pathway may have beneficial therapeutic effects for patients with Type 2 diabetes. Furthermore, an additional benefit to targeting Rap1 signaling is the potentiation of insulin secretion, which could possibly prevent or reverse β-cell dysfunction (i.e., defects in both β-cell mass and insulin secretory capacity) in diabetes.