An integrative approach to investigate treatment-resistant lung adenocarcinoma Open Access

Rackley, Briana (Spring 2020)

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Lung cancer is the leading cause of cancer-related deaths, killing 135,720 people per year. Co-mutation of the oncogene KRAS and the tumor suppressor LKB1 has been shown to increase disease severity, promote metastasis, and decrease survival. The impact of these co-mutations on patient outcomes has been well studied, but how these two mutations work together to promote tumorigenesis is unknown. The work in this dissertation uses in vivo models and patient data to demonstrate that synergy between KRAS and LKB1 is driven by autonomous growth, proliferation, and co-activation of downstream targets. Additionally, tumorigenesis is dependent upon high levels of oncogenic KRAS. Using Drosophila melanogaster, we determined that knockdown of Lkb1 works with RasV12 to override organ size control. This increase in organ size was driven by autonomous proliferation and offset by autonomous cell death. Additionally, RasV12 and Lkb1 knockdown work together to promote filamentous actin disorganization and basement membrane degradation. To further elucidate the mechanisms by which oncogenic KRAS and loss of LKB1 promote tumor progression and impede treatment response, we sought to understand how levels of oncogenic Ras contribute to Lkb1-null tumor progression and uncover novel signaling pathway components that may be targetable therapeutically. Comparison of high RasV12 expression (RasHi) to low/moderate RasV12 expression (RasLo) shows that RasHi is required for complete neoplastic transformation of Lkb1-null tissues. The effects of RasHi extend beyond tumor initiation, as RasHi levels drive tumor progression and metastasis via breakdown of basement membrane and collagen structures resulting in dissemination into secondary sites. We show that phenotypes observed using Drosophila are also observed in human patients, as co-mutation of high levels of KRAS and loss of LKB1 were shown to decrease overall patient survival compared to low level KRAS expression. Finally, we determined that tumor severity is likely driven by unprecedented co-activation of AMPK and mTOR signaling, promoting cell autophagic mechanisms and unrestricted growth. Indirect inhibition of AMPK via the CaMKII inhibitor KN-93 was shown to partially rescue observed phenotypes, offering potential avenues for continued exploration. Follow-up studies in this area will help in providing opportunities for better treatment of this subset of patients.

Table of Contents

Table of Contents

Chapter 1. Introduction                                                                                              Pages 1-38            

           1.1     Lung cancer

                      1.1.1  Lung cancer overview                                                                              2-3

                      1.1.2  Lung cancer subtypes                                                                                  4

                      1.1.3  Common lung cancer mutations                                                           5-6

                      1.1.4  Current treatments                                                                                 7-10

                      1.1.5  Racial and ethnic disparities                                                              10-12

1.2     KRAS background

                      1.2.1  RAS superfamily                                                                                          13

                      1.2.2  KRAS in cancer                                                                                      14-15

                      1.2.3   KRAS prognosis and treatment in lung cancer                               16-17

                      1.2.4   mTOR activation in KRAS-mutant lung cancer                              17-18


1.3     LKB1 background

                      1.3.1  LKB1 in disease development                                                             18-20

                      1.3.2  LKB1 as a master regulator                                                                   21-22

                      1.3.3   Regulation and function of AMPK                                                           23

                      1.3.4  KRAS and LKB1 in lung cancer                                                           24-26


1.4     Cancer cell metastasis

                      1.4.1  Metastasis overview                                                                               27-28

                      1.4.2  Metastatic heterogeneity                                                                      29-30

                      1.4.3   Metastasis in lung cancer                                                                     30-31

           1.5     Drosophila melanogaster

                      1.5.1  Drosophila melanogaster as a model organism                                31-32

                      1.5.2   Drosophila melanogaster biology                                                             32

                      1.5.3   Drosophila melanogaster genetics                                                      32-35

                      1.5.4   Understanding organ size control using Drosophila                       36-37

                      1.5.5   Drosophila melanogaster as a model for human cancer                       37


1.6      Rationale and scope of dissertation                                                                37-38

Chapter 2. Oncogenic Ras cooperates with knockdown of the tumor suppressor      39-64

Lkb1 by RNAi to override organ size limits in Drosophila wing tissue

2.1     Introduction                                                                                                        41-43

2.2     Methods                                                                                                              44-47

2.3     Results                                                                                                                48-58

2.4     Discussion                                                                                                          59-61

Chapter 3. The levels of oncogenic Ras control clonal growth dynamics to              65-106

transform Lkb1-mutant tissue in vivo

3.1     Introduction                                                                                                        67-69

3.2     Methods                                                                                                              70-79

3.3     Results                                                                                                               80-99

3.4     Discussion                                                                                                        100-102

Chapter 4. Summary and Future Directions                                                                   107-124


4.1      Discussion of dissertation                                                                               108-109

4.2      Oncogenic RasV12 drives Lkb1-mutant tissue overgrowth                      109-112

4.3      High levels of oncogenic RasV12 are required for neoplastic                         112-117

transformation and metastatic spread


References                                                                                                                            118-131

List of Figures


Figure 1.1. Leading sites of estimated new cancer deaths – 2020.                             Page 3

Figure 1.2. Oncogenic driver mutations in early stage lung adenocarcinoma.        Page 6

Figure 1.3. Racial disparities in cancer survival rates.                                                  Page 12

Figure 1.4. KRAS mutations by cancer type.                                                                   Page 15

Figure 1.5. LKB1 mutations by cancer type.                                                                   Page 20

Figure 1.6. LKB1 functions in biological processes.                                                      Page 22

Figure 1.7. Co-mutation of KRAS and LKB1 drives decreased progression-free     Page 26


Figure 1.8. Overview of metastatic progression.                                                           Page 28


Figure 1.9. Understanding Drosophila biology and genetics.                                      Page 35


Figure 2.1. RasV12/Lkb1RNAi mutations override 3rd instar wing imaginal disc Page 50

size control.


Figure 2.2. Co-mutant RasV12/Lkb1RNAi overrides 3rd instar eye imaginal disc      Page 52

size control.


Figure 2.3. Expression of co-mutant RasV12/Lkb1RNAi drives autonomous cell      Page 55

proliferation and autonomous cell death.


Figure 2.4. RasV12 promotes basement membrane degradation of Lkb1-mutant   Page 58


Figure S2.1. Co-mutation of RasV12 with loss of Lkb1 function causes adult Page 62

wing overgrowth.


Figure S2.2. RasV12/Lkb1RNAi rescue 3rd instar wing imaginal disc cell size.           Page 63

Figure S2.3. Co-mutant RasV12/Lkb1RNAi drives F-actin filament               Page 64


Figure 3.1. Clonal loss of Lkb1 in vivo results in autonomous cell death.               Page 82


Figure 3.2. Oncogenic RasHi promotes the malignant transformation of Lkb1      Page 85

mutant tissue.


Figure 3.3. SiMView light sheet microscopy allows visualization of local and      Page 89

distant collagen IV degradation by tumor cells over time. 


Figure 3.4. RasHi/Lkb1-/- mutant cells exhibit single and multi-cell dynamics       Page 91

during cell migration in vivo.


Figure 3.5. Oncogenic RasHi promotes co-activation of AMPK and mTOR in        Page 94

Lkb1-mutant malignant tumors in vivo.


Figure 3.6. High level oncogenic KRAS drives decreased patient survival and   Page 98

is associated with AMPK activation in LKB1 mutant patients.


Figure S3.1. Blocking cell death with P35 in Lkb1 mutant clones does not           Page 103

phenocopy RasLo/ Lkb1-/-.


Figure S3.2. High level oncogenic Ras promotes proliferation and S-phase         Page 104

progression of Lkb1-mutant tissue.


Figure S3.3. Acidic vesicle accumulation in RasHi/Lkb1-/- tissue.                            Page 105


Figure S3.4. High level KRAS does not result in survival differences in TP53      Page 106

mutant lung cancer patients.

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