Author ORCID Identifier

0000-0002-9473-5926

Date of Graduation

12-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Peter Friedl

Committee Member

Gary Gallick

Committee Member

Tomasz Zal

Committee Member

Pierre McCrea

Committee Member

Menashe Bar-Eli

Abstract

ABSTRACT

HYPOXIA-INDUCED AMOEBOID CANCER CELL MIGRATION: PRINCIPLES, MOLECULAR MECHANISMS AND PLASTICITY

Veronika te Boekhorst, M.S.

Supervisory Professor: Peter Friedl, Ph.D.

Cancer invasion and metastasis can occur through single cell and collective strategies with different consequences for cell and tissue patterning and metastatic outcome. Tumor hypoxia, by elevating hypoxia-inducible factors (HIF), is an established inducer of metabolic and invasive reprogramming of cancer cells, such as the induction the epithelial to-mesenchymal transition (EMT). However, how hypoxia/HIF impacts on established epithelial cancer cell migration programs and which mechano-signaling adaptations are induced upon hypoxic stress, remains unknown.

Using epithelial breast cancer (BCC) and head and neck squamous carcinoma (HN-SCC) spheroids in 3D fibrillar collagen, we characterized the invasion patterns as well as mechano-signaling requirements of hypoxia-induced cancer cell migration modes. While epithelial cancer cells migrated collectively under normoxic conditions, hypoxia (0.2% O2) or pharmacological stabilization of HIF-1α by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG), induced the transition of collective-to-single cell migration. Besides mesenchymal-like movement, most epithelial cells converted to amoeboid migration with distinctive blebby protrusions or, less frequently, with actin-rich pseudopodal protrusions towards the direction of migration. Both bleb-based and pseudopodial protrusions supported polarized interactions with collagen fibrils, allowing amoeboid cells to move equally fast (0.1-0.5 µm/min) and persistently (confinement ratio: 0.1-0.6) as elongated, mesenchymal-like single cell migration modes. HIF-induced blebby amoeboid migration was dependent on (i) RhoA/ROCK mediated actomyosin contraction for efficient bleb formation and migration, (ii) reduced β1 integrin-mediated matrix interactions, and (iii) independent on matrix-metalloproteinase-mediated collagen remodeling.

As central mechanism underlying phenotypic conversion towards blebby amoeboid migration, HIF stabilization upon hypoxia or DMOG treatment upregulated calpain-2 expression and enzymatic activity, which, by cleavage of focal adhesion protein talin-1, limited β1 integrin function, which was rate-limiting for the adaptation to and maintenance of amoeboid movement. Accordingly, interference with calpain function by pharmacological inhibitors or RNA interference prevented talin-1 cleavage, restored β1 integrin surface activity, downstream Src signaling, and reverted blebby amoeboid migrating cells towards spindle-shaped, mesenchymal-like phenotypes. Likewise, ectopic expression of a calpain-uncleavable talin protein restored active β1 integrin expression and converted amoeboid blebby migrating cells towards elongated migration morphologies.

Consistent with elevated calpain activity observed during 3D spheroid collagen migration in vitro, calpain activity was elevated in roundish, amoeboid disseminating single cells observed at the tumor-stroma interface in human HN-SCC tumor xenografts models. Calpain function was further required for round blebby amoeboid migration after HIF-stabilization in vivo.

Together, our data establish HIF signaling as microenvironmentally regulated pathway for cell reprogramming which induces blebby amoeboid migration in cancer cells. Our data further identify calpain-2 as master regulator upstream of β1 integrin shutdown, via talin-1 cleavage, which induces and maintains the conversion between elongated and amoeboid tumor cell dissemination. The HIF induced amoeboid migration program represents a migratory escape strategy in epithelial tumor cells to abandon metabolically perturbed tissue regions, and drives molecular and functional plasticity of cancer cell dissemination.

Keywords

HIF, amoeboid migration, bleb protrusions, calpain, integrin, phenotypic switching

Available for download on Thursday, August 16, 2018

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