Characterization of Protein Kinase Signaling in Cancer Metastasis and Cancer Drug Resistance Open Access

Alesi, Gina Nicole (2016)

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Despite improvements in cancer therapy and patient survival, there were approximately 589,000 human cancer deaths in 2015. Cancer metastases that are resistant to current therapeutic strategies are responsible for over 90% of these deaths. Over the past 20 years, the paradigm of cancer treatment has shifted toward targeted therapies, which act on cancer-specific molecular targets, such as kinases, to inhibit cancer cell survival and growth. We recently exploited two key strategies for determining novel protein kinase targets in cancer therapy. The first strategy relies on identifying signaling effectors downstream of oncogenic kinases. Following previous research demonstrating that ribosomal S6 kinase 2 (RSK2) promotes head and neck cancer cell invasion and tumor metastasis, we performed phospho-proteomics studies to identify RSK2 signaling effectors in metastasis. Among top candidates were several potential targets involved in cell migration and cytoskeletal regulation. We validate and characterize stathmin, a novel RSK2 substrate and cytoskeletal regulatory protein that promotes microtubule destabilization. We demonstrate that RSK2 regulates microtubule stability to provide a morphological advantage for promoting cancer cell invasion and tumor metastasis, which is partly mediated through the phosphorylation and inhibition of stathmin. The second strategy relies on the principle of synthetic lethality in cancer therapy, in which targeting a synthetic lethal gene to a chemotherapy drug could potentially increase therapeutic efficacy and decrease off-target side effects. We performed a screen for kinases that promote chemosensitization when targeted in combination with cisplatin. Genetic depletion of the top screening candidate, microtubule associated serine/threonine kinase 1 (MAST1), sensitizes various cancer cells to sub-lethal doses of cisplatin. Additional phospho-antibody array and mass spectrometry studies reveal that MAST1 signals through several protein effectors, including MEK1 and PLK1, to promote cell survival and proliferation during cisplatin treatment. Collectively, these studies expand our understanding of protein kinase signaling mechanisms underlying cancer cell metastasis and drug resistance.

Table of Contents

Abstract ii

Table of Contents iv

List of Figures vi

List of Abbreviations viii

Chapter 1: Introduction 1

1.1 Kinases 1

1.2 Mechanistic Basis of Protein Kinase Signaling 1

1.3 Cancer and the Kinome 3

1.4 Targeting Protein Kinases in Cancer 6

1.5 Clinical Challenges of Cancer Therapy 8

1.5.1 Cancer Metastasis 8

1.5.2 Drug Resistance 11

1.6 Identifying Novel Protein Kinase Therapeutic Targets for Cancer Therapy 14

Chapter 2: RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis 16

2.1 Author's Contribution and Acknowledgement of Reproduction 16

2.2 Abstract 17

2.3 Introduction 18

2.4 Materials and Methods 21

2.5 Results 28

2.6 Discussion 45

2.7 Acknowledgements 48

Chapter 3: Microtubule-associated serine/threonine kinase 1 (MAST1) kinase activity is important for cisplatin resistance in human cancers 49

3.1 Abstract 49

3.2 Introduction 50

3.3 Materials and Methods 53

3.4 Results 60

3.5 Discussion 95

3.6 Acknowledgements 98

Chapter 4: General Discussion and Future Directions 99

4.1 RSK2 and Cancer Metastasis 99

4.2 MAST1 and Therapeutic Resistance 102

4.3 Conclusions 105

Chapter 5: References 107

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