| dc.description.abstract | Although under active investigation, alterations within the microenvironment that are causative to, and a consequence of, tumor progression are only beginning to be better appreciated through advances in imaging, experimental, and theoretical tools. The tumor microenvironment can be broadly classified into acellular components - the extracellular matrix (ECM) and cellular components, which though not transformed, may exhibit altered phenotypic traits upon interactions with cancer cells. The ECM, once thought to be a passive scaffold of tissues exerts both mechanical-physical and biochemical effects on cells, which in turn remodel it. Herein, I will showcase novel observations from my dissertation research that pertain to the transduction of extracellular cues from the microenvironment with potent effects on breast cancer cell migration.
Within the tumor microenvironment, stromal cells such as fibroblasts play complex multidimensional roles in cancer invasion and metastasis. Fibroblasts, originally resident within the untransformed tissues (normal fibroblasts or NF) are known to impede growth and migration of cancer cells. In contrast, after exposure to tumor- derived signals, they can be modified or replaced by a population with altered traits (now known as cancer-associated fibroblasts or CAFs) whose functions could be opposite to their normal counterparts. Several studies demonstrate invasion- promoting functions of CAFs. How CAFs differ from NFs especially in communicating cues to, and regulating traits of, cancer cells remain relatively ill-understood. The elucidated mechanisms of such roles include remodeling and realigning of the surrounding and confining ECM such as fibrillar Collagen and Fibronectin leading to enhanced migration. However, CAFs also secrete ECM, which in specific contexts has been proposed to confine and restrict tumor growth. The identification of novel molecular signatures that are cognate to invasion-promoting CAF niche is pertinent to recent translational efforts that seek to harness this information to target malignant tumors.
In the first part of my PhD research, I constituted time-lapse-tractable 3D pathotypic cultures comprising breast cancer cell lines with fibroblasts enriched from invasive ductal carcinoma biopsies. Herein, CAFs enhanced breast cancer migration relative to NFs, which was also supported through greater orthotopically injected cancer cell infiltration within murine inguinal lymph nodes. The enhancement was also seen in cancer cells exposed to CAF-secreted medium, which also phenocopied laminin-rich matrix in compacting cancer clusters. A subsequent immunocytochemical screen identified laminins α5, -β1 and -γ1 to be highly expressed in enriched CAFs populations as well as in stromal areas of breast cancer sections. Depleting laminin- α5, -β1, and -γ1 in CAFs downregulated 3D cancer cell migration. Cancer cells cultivated on Laminin-511 substrata showed increased adhesion, faster and persistent migration, enhanced shape polarization, and deformability of cancer cells relative to Laminin-211 controls. Observation of higher invadopodial F-actin branching dynamics on Laminin-511 led me to assay for and demonstrate anisotropically corticalized Arp 2/3 in cancer cells. An integrin antibody screen showed Integrin α6β1 inhibition specifically nullified Laminin-511-driven enhancement of morphomigrational traits of cancer cells, like Arp2/3 inhibition. Moreover, cells on Laminin-511 showed enhanced Integrin α6 localization to their cortices. These results lead me to propose that activated fibroblasts use laminin-511 to localize cognate integrin receptors, and Arp2/3 of cancer cells to their cortices, resulting in higher Arp2/3-driven actin remodeling and enhanced cell migration. These findings combined with my demonstration of greater glycolytic and citric acid pathway flux suggest a microenvironmental regulation of the activity of cancer cells. Notwithstanding the transduction of biochemical cues emanating from CAF-secreted ECM, biophysical cues from the ECM such as matrix stiffness also regulate cancer progression. In the second part of my PhD research, I aimed to decipher the role of matrix stiffness in regulating cancer migration through dynamical alterations in metabolic fluxes within cells.
Most solid tumors are found to be stiffer than their normal counterparts. One of the causes of breast cancer tissue stiffening is matrix stiffening, which is due to both matrix crosslinking and fresh matrix deposition. A balance between matrix degradation and deposition is required for maintaining homeostasis; disruption of this homeostasis is a hallmark of invasive tumors. The stiffening of matrix surrounding the cancer cells regulates proliferation, tumorigenesis, drug resistance, stemness, immune evasion, invasion, EMT and metastasis. On the other hand, alteration in cancer metabolism is also an emerging hallmark of cancer. Although Otto Warburg observed an increased dependence of cancer cells on aerobic glycolysis (known as the Warburg effect), current evidence suggest that cancer cells can show higher activity of both glycolysis and mitochondrial oxidative phosphorylation in a context-dependent manner. Recent reports indicate that invasive cancer cells exhibit distinct morphomigrational and metabolic traits when confronted with biophysically variant matrix microenvironments enroute metastasis. Whether dynamical shifts in such traits are interlinked through a common molecular program remains elusive. Using triple negative breast cancer cell lines on Collagen I substrata-coated hydrogels recapitulating stiffness values of non- cancerous breast tissue and the desmoplasia of tumors, I observed greater cell shape polarization and migration in the latter.
Associated lower lactate and pyruvate levels in such conditions motivated me to examine their pyruvate kinase M2 expression, which showed nuclear and cytoplasmic localization in softer and stiffer environments respectively. Pharmacologically impairing PKM2 activity in stiffer substrata decreased migration and shape polarization of cancer cells while increasing lactate and pyruvate levels. In contrast, increasing its activity on softer substrata attenuated cancer cell migration and elongation. I assayed for localization of the mechanosensory protein YAP upon PKM2 activity modulation: PKM2 activation increased nuclear YAP localization on soft substrata. Pharmacologically inhibiting YAP on stiff substrata not just decreased migration but also increased nuclear localization of PKM2 and lactate and pyruvate levels. A predominantly cytoplasmic localization of PKM2 was confirmed in biopsy sections of patients with invasive ductal breast cancer. I propose that a reciprocal repartitioning of PKM2-YAP interlinks the cognate metabolic and migrational states of cancer cells; targeting such positive feedback may hold the key to future therapeutic strategies.
In another short project, evidence is presented for linked heterogeneities in α2,6-linked sialic acid expression and metabolism in breast cancer. Phenotypic heterogeneity and aberrant glycan expression on the cell surface are two prominent hallmarks of cancer. Our previous results identify differential levels of a specific glycan linkage: α2,6-linked sialic acids within breast cancer cells in vivo and in culture due to differential expression of the glycosyltransferase ST6GAL1. The triple negative breast cancer cell line MDA-MB-231 consists of a moderate α2,6-linked sialic acid expressing cell niche (“moderate”), which migrates more prolifically through pathotypic extracellular matrices than the counterpart high α2,6-linked sialic acid expressing cell niche (“high”). Here, we show a spatial congruence between heterogeneity in sialic acid expression and glucose metabolism. High cells exhibit greater glucose uptake than moderate cells. High cells also show a higher expression of the glucose transporter GLUT3 and higher rate of ATP production from the glycolytic pathway. Inhibiting glucose flux using 2-Deoxyglucose (2-DG) in high cells enhanced their invasion through complex 3D matrix microenvironments. Finally, stably knocking down ST6GAL1 in the high 2,6- Sial cells also decreases glucose uptake suggesting an association between 2,6-sialic acid expression and glucose uptake drives breast cancer migration.
Altogether, these findings reveal novel roles for both cellular and acellular components of the tumor microenvironment in driving cancer progression, paving the way for innovative therapeutic strategies that disrupt tumor progression by targeting the interplay between matrix cues, metabolism, and migration. | en_US |
