K811 acetylation

K811 acetylation regulates ZEB1 dimerization, protein stability, and NuRD complex interactions to promote lung adenocarcinoma progression and metastasis Mabel G. Perez-Oquendo, Roxsan Manshouri, Ph.D., Jared J. Fradette, Don L. Gibbons, M.D., Ph.D. University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Department of Thoracic Head & Neck Medical Oncology 4A 4B Ruth L. Kirschstein National Research Service Award Individual Pre-doctoral Fellowship to Promote Diversity in Health-Related Research (NCI 1F31CA268343-01). M.P.O. is supported by the Research Supplements to Promote Diversity in Health-Related Research (NCI R37CA214609-03).

Contact info: MGPerez2@mdanderson.org

Introduction Lung cancer is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into invasive mesenchymal cells. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple groups have reported discrepancies in the observed molecular weight (~ 190- 250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in a panel of lung adenocarcinoma cell lines. We aim to characterize a novel regulatory mechanism of the transcriptional repressor ZEB1 with the goal of identifying its functional and pathological relevance to the metastatic process. Lung Cancer is the leading cause of cancer-related deaths Males 319,420 Females 289,150 Lung & bronchus Prostate Colon & rectum 21% 11% 9% Lung & bronchus Breast Colon & rectum 21% 15% 8%

K811 acetylation site is a novel PTM in ZEB1 identified by mass spectrometry

Dimerization facilitates ZEB1/NuRD complex interaction and binding at the promoter of its target genes

Co-Immunoprecipitation

8. A.

Hypothesis ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis Figure 5. Schematic diagram of the PTMs in ZEB1 identified by tandem-mass spectrometry (MS/MS) analysis: T131, Y577/Y578, S624, S657, K811. The circles indicate phosphorylation (P), and the diamond indicates acetylation (Ac).

Proximity Ligation Assay

8. B.

GFP-ZEB1/HDAC1

GFP

Zeb1 (WT)

Zeb1 (K811R)

Zeb1(K811Q)

✱✱✱✱

Zeb1 (K811R)

Zeb1 (K811Q)

Zeb1 (WT)

GFP

GFP ZEB1_WZETB1_K8Z1E1BR1_K811Q

Results

GFP-ZEB1/CHD4

K811 acetylation regulates ZEB1 dimerization and protein stability

✱✱✱✱

6. A.

6. B.

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Figure 1. 2022 cancer statistics for cancer related deaths in USA.

Siegel et al., 2022

Acetyl- deficient

Acetyl- mimetic

Mouse model of metastatic lung adenocarcinoma

Zeb1 (K811R)

Zeb1 (K811Q)

Zeb1 (WT)

GFP

GFP ZEB1_WZETB1_K8Z1E1BR1_K811Q

8. C.

ZEB1

CHD4

✱✱

✱✱✱

1.5

Zeb1 (WT) Zeb1 (K811Q) Zeb1 (K811R)

GFP Zeb1 (WT)

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✱

GFP Zeb1 (WT)

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0 2 4 6 8 10

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Zeb1 (K811R) Zeb1 (K811Q)

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1.0

Zeb1 (K811R) Zeb1 (K811Q)

✱✱

✱✱✱

✱✱

0.5

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0.0

4 8 16

Time (hrs)

miR200c-141 SEMA3F

N-Myc

GAPDH

miR200c-141 SEMA3F

N-Myc

GAPDH

Figure 6. (A) GFP-vector, GFP-ZEB1, GFP-ZEB1_K811R (acetyl-deficient), and GFP-ZEB1_K811Q (acetyl-mimetic) were co-transfected into 393P cells and induced with doxycycline for 24 h. Blots for GFP, ZEB1 and β -actin confirm that ZEB1 WT and acetyl-mimetic mutant are a 250 kDa dimer while the acetyl-deficient mutant is a 125 kDa monomer. (B) GFP-ZEB1 acetylation mutant and control 393P cells were treated with 100 µg/ml cycloheximide (CHX), collected for the indicated time point, and immunoblotted with antibodies against GFP and β - actin. Densities of ZEB1 bands were measured using ImageJ software and normalized to the expression of actin (loading control). The value of normalized ZEB1 at time 0 was set at 1.0. Blots and graphs confirm that 250 kDa ZEB1 band prolongs half-life.

Figure 8. (A) GFP-ZEB1 acetylation mutant and control 393P cells were co-immunoprecipitated with antibodies against mouse IgG, GFP, and HDAC1, and immunoblotted with antibodies against GFP, MTA1, HDAC1, and β -actin. Blots confirm that ZEB1 dimers interact with the NuRD complex. (B) GFP-ZEB1 acetylation mutant and control 393P cells were probed with ZEB1 and NuRD complex members antibodies using the Duolink proximity ligation assay (PLA). Images and graphs confirm the ZEB1 dimer/NuRD interaction in situ. (C) ChIP with antibodies against ZEB1, CHD4 and control IgG in GFP-ZEB1 acetylation mutant and control 393P cells. ****P < 0.0001; ***P < 0.005; **P < 0.001; *P < 0.05.

Figure 2. The genetically engineered mouse model (GEMM) acquires the somatic activation of the Kras G12D allele to develop spontaneous lung adenocarcinoma, without metastatic formation. Introduction of a mutant p53 R172H Δ G allelewas found to recapitulate features of metastasis-prone lung adenocarcinoma cancer patients. The derived cell lines from this GEMM were subcutaneously injected into syngeneic mice to evaluate the propensity to metastasize. The cell line 393P was defined as a metastasis incompetent cell line, and 344SQ was a metastasis prone cell line. ZEB1 transcriptional repressor regulates Epithelial-to-mesenchymal transition (EMT)-contributed to lung cancer metastasis

K811 acetylation promotes NSCLC invasion in vitro and metastasis in vivo Mean and Error

K811 acetylation protects ZEB1 from proteasomal degradation by the action of the E3 Ubiquitin Ligase (UBL) SIAH1

7. A.

5% input

IgG

IP: GFP

IP: Ubiquitin

9. D.

9. B.

9. C.

9. A.

✱✱✱

GFP

Mesenchymal

Migration

Invasion

Epithelial

2000

WT R Q -

WT R Q +

WT R Q -

WT R Q +

WT R Q -

WT R Q +

WT R Q -

WT R Q +

GFP Zeb1 (WT)

MG132 (10μM):

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n.s.

Epithelial Cells: High cell-cell contact Low motility and invasiveness Increased E-cadherin level

Zeb1 (WT)

1500

GFP

Zeb1 (WT)

GFP

Zeb1 (K811R) Zeb1 (K811Q)

✱✱✱

1000

102 kDa 225 kDa 150 kDa

Doxycycline

500

Ubiquitin

Zeb1 (K811R)

Zeb1 (K811Q)

GFP Zeb1 (WT)

75 kDa 50 kDa

Time (days)

GFP ZEB1 (WZET)B1 (K811R) ZEB1 (K811Q)

Zeb1 (K811R)

Zeb1 (K811Q)

9. E.

GFP

Zeb1 (WT)

Zeb1 (K811R)

37 kDa

102 kDa 225 kDa 150 kDa

Mesenchymal Cells: Low cell-cell contact High motility and invasiveness Increased expression of EMT transcription factors GFP-ZEB1 β-actin 7. B.

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Transcription Factors ZEB1

Zeb1 (K811R)

Zeb1 (K811Q)

GFP Zeb1 (WT)

Zeb1 (WT)

Zeb1 (K811R)

Zeb1 (K811Q)

7. C.

Repression of epithelial state

Induction of mesenchymal state

GFP - + - + - + - +

Zeb1 (K811R) Zeb1 (K811Q)

0 1000 2000 3000 4000 5000

✱✱ ✱✱

✱✱ ✱✱✱

20 40 60 80

SIAH1A-KD:

102 kDa 225 kDa 150 kDa

EMT

GFP-ZEB1

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10 15

GFP Zeb1 (WT)

Zeb1 (K811R)

Zeb1 (K811Q)

GFP Zeb1 (WT)

Zeb1 (K811R)

Zeb1 (K811Q)

✱✱

SIAH1A

0 5

Figure 3. Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis that results in a loss of apical-basal polarity and epithelial cell-cell contacts to acquire mesenchymal front-back polarity that confers migration and invasion. The Zinc finger E-box binding homeobox 1 (ZEB1) transcriptional repressor is a master regulator of EMT.

Summary and working model • K811 acetylation regulates ZEB1 dimerization and protein stability. • Acetylation protects ZEB1 from proteasomal degradation by the action of the UBL SIAH1. • Dimerization facilitates recruitment of the NuRD complex to genomic promoters. • ZEB1_K811ac promotes NSCLC metastasis via EMT. Figure 9. GFP-ZEB1 acetylation mutant and control 393P cells were seeded and induced with doxycycline in 24-well (A) chemotaxis or (B) haptotaxis chambers. (C) GFP-ZEB1 acetylation mutant and control 344SQ cells were subcutaneously implanted into the flank of syngeneic WT 129Sv mice. The growth of the primary tumor was measured. Once tumors reached a volume of 100 mm 3 , we gave the mice doxycycline, and when the tumors reached 1500 mm 3 , we killed the mice via CO 2 . (D) Metastatic lung nodules were quantified and (E) validated by hematoxylin and eosin staining of paraffin-embedded lung tissue.

- + Zeb1 (K811Q)

- +

- + GFP

GFP (24 kDa)

SIAH1A-KD:

Zeb1 (WT)

Zeb1 (K811R)

β-actin

SIAH1A

ZEB1

250 kDa ZEB1 is an SDS-PAGE-resistant homodimer

7. D.

Zeb1 (WT)

Zeb1 (K811Q)

Zeb1 (K811R)

4. A.

4. B.

SIAH1A-KD:

0 12 24

0 12 24 hrs

CHX (100 μg/mL):

102 kDa 225 kDa 150 kDa

GFP-ZEB1

SIAH1A

dimer

β-actin

1.5

siSIAH1A scramble

1.0

monomer

0.5

0.0

Time (hrs)

Figure 7. (A) GFP-ZEB1 acetylation mutant and control 393P cells were treated with 10 µM proteasome inhibitor MG132 for 8h. The whole- cell lysate was co-immunoprecipitated with antibodies against mouse IgG, GFP, and ubiquitin, and immunoblotted with antibodies against ubiquitin, GFP, and β -actin. Input indicates 5% whole cell lysate. Blots for ubiquitin antibody confirm greater ZEB1 ubiquitination in cells expressing the acetyl-deficient mutant. qPCR (B) and immunoblotting (C) of SIAH1 and ZEB1 in GFP-ZEB1 acetylation mutant and control 393P cells transfected with Dharmacon SMARTpool siRNAs for SIAH1. (D) GFP-ZEB1 acetylation mutant and control 393P cells transfected with siSIAH1 were treated with 100 µg/ml CHX, collected for the indicated time point, and immunoblotted with antibodies against GFP, SIAH1, and β -actin. Densities of ZEB1 bands were measured using ImageJ software and normalized to the expression of actin (loading control). Blots and graphs confirm that silencing SIAH1 upregulates the ZEB1 protein expression and stability.

Figure 4. (A) Immunoblotting of ZEB1 and β -actin in non-metastatic 393P and metastatic 344SQ murine cell lines. Blot confirms the presence of both 125 kDa and 225 kDa ZEB1 bands. (B) GFP-vector alone, GFP-vector plus FLAG-ZEB1, GFP- ZEB1 alone, or GFP-ZEB1 plus FLAG-ZEB1 were co-transfected into 344SQ cells and induced with doxycycline for 24 h. The whole-cell lysate was co-immunoprecipitated with antibodies against mouse IgG and GFP, and immunoblotted with antibodies against Flag, GFP, and β -actin. Blot for Flag antibody confirms that GFP-ZEB1 co-immunoprecipitates with FLAG-ZEB1.

Acknowledgements This research is funded by the Ruth L. Kirschstein National Research Service Award Individual Pre-doctoral Fellowship to Promote Diversity in Health-Related Research (NCI 1F31CA268343-01). M. Perez-Oquendo acknowledges Drs. Gibbons and Barton, as well as her advisory committee for their continued mentorship and support throughout this project .

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