Identifying oncogenic attributes and protein binding partners of the NKX2-1 lung cancer oncogene

Sushmita Sridhar May 2014

ii

IDENTIFYING ONCOGENIC ATTRIBUTES AND PROTEIN BINDING PARTNERS OF THE NKX2-1 LUNG CANCER ONCOGENE

An Honors Thesis Submitted to

the Department of Biology in partial fulfillment of the Honors Program

STANFORD UNIVERSITY

by SUSHMITA SRIDHAR

MAY 2014

iii

iv

There are several people and organizations without whom this research and thesis would not have been possible. Thank you to the Pollack lab for research advice and support, particularly to Dr. Pollack for help in designing experiments and providing invaluable input on my thesis, Dr. Mary Beth Mudgett for reading my thesis, Dr. Russ Carpenter for providing thesis writing support through the course Bio 199W, Stanford Undergraduate Research and Advising (UAR) and the Vice Provost for Undergraduate Education for funding support to conduct summer research.

v

Table of Contents ABSTRACT .................................................................................................................................... 1

INTRODUCTION .......................................................................................................................... 2

MATERIALS AND METHODS................................................................................................... 6 CELL LINES ...................................................................................................................................................6 NKX2-1 PLASMID CONSTRUCTS ...................................................................................................................6 DNA TRANSFECTIONS ..................................................................................................................................6 LENTIVIRAL INFECTIONS...............................................................................................................................7 CELL LYSATE PREPARATION FOR CO-IPS ......................................................................................................7 CROSS-LINKING EXPERIMENTS .....................................................................................................................8 MAGNETIC BEAD PREPARATION....................................................................................................................8 IMMUNOPRECIPITATIONS ..............................................................................................................................9 IMMUNOBLOTTING........................................................................................................................................9 SILVER STAINING .......................................................................................................................................10 MASS SPECTROMETRIC ANALYSIS...............................................................................................................10

RESULTS...................................................................................................................................... 11 NKX2-1 ISOFORMS IN LUNG CANCER CELL LINES HCC 1195 AND HCC 1833 ...........................................11 EVALUATING THE ONCOGENIC POTENTIAL OF NKX2-1 ISOFORMS IN LUNG CANCER .................................12 PURIFYING NKX2-1 BINDING PARTNERS BY CO-IMMUNOPRECIPITATION ..................................................12 MASS SPECTROMETRY ANALYSIS OF CO-IMMUNOPRECIPATED PROTEINS...................................................14 OPTIMIZATION OF CO-IMMUNOPRECIPITATION CONDITIONS.......................................................................15 CROSS-LINKING PROTEINS TO PRESERVE PROTEIN INTERACTIONS WITH NKX2-1......................................16

DISCUSSION................................................................................................................................ 18

BIBLIOGRAPHY......................................................................................................................... 22  

vi

List of tables Table 1. Mass spectrometry analysis to identify potential protein binding partners of NKX2-1.

vii

List of figures Figure 1. Evaluating NKX2-1 isoform expression in lung cancer cells. Figure 2. Co-immunoprecipitation of NKX2-1 and known binding partner. Figure 3. Mass spectrometry analysis of NKX2-1 co-immunoprecipitates. Figure 4. Comparison of two co-immunoprecipitation protocols and conditions to eliminate non-specific binding. Figure 5. Cross-linking proteins in cultured cells and subsequent immunoprecipitation.

1

Abstract

Lung cancer is the leading cause of cancer death in the United States, and the

oncogene NXK2-1 has been previously been implicated in a subset of lung cancers.

NKX2-1 encodes a transcription factor known to be important in controlling normal lung

development. However, NKX2-1 is also found to be amplified and overexpressed at the

DNA and protein levels, respectively, in approximately 15% of lung cancers, where it

contributes to abnormal cell proliferation and survival, and thus it is classified as an

oncogene. Although it is presumed that NKX2-1 must be behaving differently in cancer

compared to normal cells, the mechanisms by which it increases cancer cell proliferation

are not well understood. Possible mechanisms include altered NKX2-1 transcriptional

activity by expression of distinct NKX2-1 protein isoforms, or by NKX2-1 association

with distinct protein binding partners. Here, we examined the phenotype of lung cancer

cells overexpressing different NKX2-1 isoforms, to better understand the protein’s

function. We also used co-immunoprecipitation to isolate proteins that associate with

NKX2-1 in lung cancer cells, and then employed mass spectrometry to identify the co-

immunoprecipitated proteins. The resulting findings provide unique and novel insight

into the mechanisms of NKX2-1 oncogenic activity, which may lead to future efforts in

targeted molecular therapy for these lung cancers.

Keywords: lung cancer, NKX2-1, co-immunoprecipitation, oncogene, protein cofactors

2

Introduction

Lung cancer is the leading cause of cancer-related death in men and women in the

United States, accounting for approximately 27% of all cancer deaths, more than prostate,

breast and colon cancer combined (Jemal et al., 2008; Society, 2008). Among lung

cancers, the most common type is non-small cell lung cancer (NSCLC), which is

composed primarily of adenocarcinomas, squamous cell carcinomas and large cell

carcinomas (Non-Small Cell Lung Cancer Treatment - National Cancer Institute; Travis

et al., 2004). Previous genomic analysis of a large collection of lung cancer cell lines and

primary tumors revealed unique amplification of chromosomal locus 14q13.3 (Kwei et

al., 2008; Kendall et al., 2007). This amplified region spans NKX2-1 (also known as TTF-

1), a 38-42 kD homeobox-family transcription factor that is essential for the normal

development and differentiation of the thyroid, central nervous system, and lungs

(Boggaram, 2009; Minoo et al., 1999; Ramirez et al., 1997; Kimura et al., 1996). In the

healthy adult lung, NKX2-1 expression is restricted to a subset of cells, specifically

alveolar type II cells and Clara cells, and it controls the expression of key lung-specific

genes, including several surfactant proteins and Clara cell secretory protein (Varma et al.,

2012; Cao et al., 2010; Boggaram, 2009; Kwei et al., 2008; Tian et al., 2006). Its

promoter in humans is controlled by HNF-3, Sp (specificity protein) 1, Sp3, HOXB3, and

GATA-6 transcription factors, and the single NKX2-1 gene is transcribed as two protein

isoform variants (with either two or three exons), although it is uncertain whether both

isoforms are expressed and functional in human lung cells (Boggaram, 2009; Shaw-

White et al., 1999; Ikeda, Clark, & Shaw-White, 1995).

3

In lung cancer cells, gene amplification of NKX2-1 leads to increased NKX2-1

RNA and protein levels, which contribute to increased lung cancer cell proliferation and

survival rates (Kwei et al., 2008). This conclusion is supported by the finding of reduced

cancer cell proliferation when endogenous NKX2-1 levels are depleted by RNA

interference (RNAi)-mediated knockdown in lung cancer cells with NKX2-1

amplification (Kwei et al., 2008). Moreover, neither lung cancer cell lines with NKX2-1

expression but without gene amplification, nor those lacking NKX2-1 expression are

affected by the NKX2-1 knockdown, thus confirming that tumor cell proliferation is

dependent upon gene amplification-driven NKX2-1 protein overexpression (Kwei et al.,

2008; Tanaka et al., 2007). The dependency on amplified expression implicates NKX2-1

as a lung cancer oncogene (Das et al., 2011; Kwei et al., 2008).

A seeming paradox and unanswered question that prior research has raised is how

NKX2-1 on the one hand controls normal lung cell differentiation, but on the other hand

can also promote lung cancer. As a transcription factor, NKX2-1 regulates the expression

of genes (Boggaram, 2009), but it is unclear in what capacity NKX2-1 acts as an

oncogene. Our overarching hypothesis is that NKX2-1 controls the expression of a

distinct set of genes in lung cancer cells (e.g. proliferation genes), compared to normal

lung cells. In exploring this hypothesis, there are several possible models that could

explain how NKX2-1 might regulate distinct genes in lung cancer. One possibility that

we investigated is whether the two different protein isoforms of NKX2-1 (long and short

forms) have different transcriptional and growth-promoting roles in lung cancer cells.

Another model that we studied is whether NKX2-1 binds to distinct protein partners (e.g.

4

transcription co-activators) in lung cancer cells, which, for example, might influence

NKX2-1 to transcriptionally activate proliferation genes.

We also recognize that alternative possibilities exist that may require exploration

in future studies. For example, it is possible that when amplified, NKX2-1 undergoes

different post-translational modifications due to an unknown set of factors, thus altering

its transcriptional activity and rendering cells cancerous. Alternatively, it is possible that

amplification of NKX2-1 occurs only in combination with another gene mutation (e.g.

TP53 mutation, not present in normal lung cells), which then together cause oncogenesis.

A third possibility is that simply higher levels of NKX2-1 expression lead to cancer.

However, this explanation seems unlikely because of research conducted in the Pollack

lab on CDX2, an analogue to NKX2-1 in colon cancers. CDX2 is a transcription factor

that regulates normal intestinal development and differentiation and whose amplification

is implicated in colorectal cancer. It was found that variation in CDX2 levels alone does

not correlate with oncogenic function (Salari et al., 2012). The similarities between

NKX2-1 and CDX2 as transcription factors would suggest that the same likely holds true

for NKX2-1.

Instead of pursuing these other hypotheses, we addressed NKX2-1’s variant

isoforms and binding partners because these are plausible scenarios based upon the

understanding of similar transcription factors implicated in cancers, and because these

possibilities can be readily tested. In addition, although it is somewhat understood how

NKX2-1 interacts with protein partners in normal lung cells, its protein interactions in

lung cancer cells have not been previously characterized, which makes this research

significant to the field. We investigated NKX2-1 binding partners by 1) co-

5

immunoprecipitating NKX2-1 and associated proteins in lung cancer cells and

subsequently identifying binding partners by mass spectrometry, 2) verifying that NKX2-

1 co-immunoprecipitates with identified proteins specifically in lung cancer compared to

normal lung cells, and 3) determining if these binding partners are essential for cancer

growth by performing RNAi knock down experiments in lung cancer cells.

Previous work in our lab, including the genomic profiling and initial functional

studies that revealed NKX2-1’s role in lung cancer, was carried out using lung cancer cell

lines that endogenously express NKX2-1 (Kwei et al., 2008). The lung cancer cell lines

chosen were determined to be appropriate because they harbor DNA amplification of

NKX2-1, express NKX2-1 at high levels, and are dependent on NKX2-1 expression for

cell proliferation and survival. Given that the interactions of interest occur at the sub-

cellular level and depend upon gene products, it was advantageous to utilize a cellular

model in order to answer questions before potentially looking into more complex

systems. By using a cell culture model, we could most accurately assess and characterize

protein interactions that are unique to lung cancer cells expressing NKX2-1.

Given that the role of NKX2-1 in promoting lung cancer has as yet proven to be

complex, our experimentation has provided greater focus for future research on the

specific NKX2-1 isoforms and protein partner interactions that we find. The limited

understanding of NKX2-1’s oncogenic characteristics and differential role in human lung

development and lung adenocarcinomas provided a framework for this research, and the

potential cancer cell-specific isoformic features and protein binding partners we

identified begin to clarify the mechanism(s) through which NXX2-1 acts as an oncogene.

6

Materials and Methods

Cell lines

HCC1195, H1819 and HCC1833 cell lines, originally obtained from the Hamon Center

for Therapeutic Oncology Research, UT Southwestern Medical Center, were cultured

(37°C, 5% CO2) in RPMI-1640 (Gibco) media supplemented with 10% fetal bovine

serum and antibiotics.

NKX2-1 plasmid constructs

NKX2-1 full-length cDNA was obtained from Origene. NKX2-1 isoform 1, encoding a

401 amino acid “long isoform”, and NKX2-1 isoform 2, encoding a 371 amino acid

“short isoform” (missing the N-terminal 30 amino acids of isoform 1) were PCR-

amplified from the NKX2-1 cDNA, sequence-verified, and then cloned into the BamHI

and XhoI sites of the pCDNA6/V5-HisC mammalian expression vector (Invitrogen).

These constructs were used for 293T cell transfections, to assist in determining the size of

the NKX2-1 isoforms expressed in lung cancer cells. NKX2-1 long and short isoforms

were also subcloned into pENTR1A (Addgene), and then recombined (by Clonase

reaction) into the pLentiCMV-Blast lentiviral expression vector (Addgene). These

constructs were used for cell transductions, for rescue experiments to determine which

isoform(s) drive proliferation of lung cancer cells.

DNA transfections

Transfections of 293T cells were carried out using Lipofectamine 2000 (Invitrogen)

according to manufacturer’s instructions. Transfections to generate lentivirus were done

7

by co-transfecting NKX2-1 constructs together with ViraPower packaging plasmids

(Invitrogen), according to the manufacturer. Viral supernatants were collected 48 hours

post-transfection, and cleared using a 45 µM filter.

Lentiviral infections

HCC1833 lung cancer cells were plated into 6-well plates at 200,000 cells per well.

Sixteen hours after plating, viral supernatant was added to each well, along with 6 µg/ml

polybrene. Cells were spin-infected at 1000 rpm for 30 minutes, repeated 4-6 hours later,

and then incubated overnight. 48 hours later, cells were re-plated into a 10 cm dish and

then subjected to 2 µg/ml of blasticidin selection for 7-14 days.

Cell lysate preparation for co-IPs

Cells were harvested at 70% confluence in 1 ml PBS by scraping. Harvested cells were

spun down at 1500 g in a centrifuge at 4°C, and the cell pellet was resuspended in 10 ml

cell membrane lysis buffer (5 mM PIPES, pH 8.0; 85 mM KCl; 1% Igepal; 1x 25x Roche

Complete Mini Protease Inhibitor Cocktail; Invitrogen Ultra-Pure water). After 15

minutes of incubation rocking at 4°C, cells were dounced 20 times in a Wheaton 7 ml

Dounce Tissue Grinder to mechanically lyse the outer cell membranes. Cell nuclei were

then centrifuged at 1500 g at 4°C and resuspended in 2 ml of nuclei lysis buffer (20 mM

Tris, pH 8; 2 mM EDTA; 2% Triton X-100; 0.2% Sodium Deoxycholate; 200 mM NaCl;

Invitrogen Ultra-Pure water). Nuclear lysates (enriched for the NKX2-1 transcription

factor) were incubated on ice for 30 minutes and then sonicated in a water bath sonicator

for 15 minutes (cycle of 30 seconds on and 30 seconds off). Sonicated nuclear lysates

8

were centrifuged at 1500 g for 15 minutes and the soluble fraction was retained and

quantified. 400 µg of lysate was added to 4.5 ml of IP Buffer (20 mM Tris, pH 8; 2 mM

EDTA; 2% Triton X-100; 0.2% Sodium Deoxycholate; 200 mM NaCl; Invitrogen Ultra-

Pure water), in addition to antibody-bound beads.

Cross-linking experiments

Cells were first exposed to the reversible cross-linking agent Dithiobis[succinimidyl

propionate] (DSP) (Zlatic et al., 2010). A 100 mM solution of DSP/DMSO was added to

warm PBS, at 10 µl DSP/DMSO per 1 ml phosphate-buffered saline (PBS). 20 ml of DSP

solution was added to a 15 cm plate of HCC1833 or H1819 cells at 80% confluence and

incubated on ice for 2-3 hours. To quench the reaction, a 20 ml solution of 40 µl 500 mM

Tris-HCl (pH 7.5) per 1 ml PBS was added to each plate of cells and incubated on ice for

15 minutes. For co-IP experiments utilizing cross-linked proteins, nuclear lysates were

prepared using the Active Motif Universal Co-IP Kit (Steps 1- 3) (Catalog No. 54002),

with subsequent IP, washing and elution steps as described below. This combination of

protocols was done to optimize co-IP stringency and reduce background.

Magnetic bead preparation

New England Biolabs Protein A (S1425S) magnetic beads were used for the co-

immunoprecipitation. 25 µl of beads (25 µg) per IP were washed in 1ml PBS before three

washes in bead blocking buffer (5 mg/ml BSA in PBS, 25x Roche Complete Mini

Protease Inhibitor Cocktail, 1x PBS). 5 µg of antibody (either Santa Cruz rabbit

polyclonal anti-TTF-1, Seven Hills rabbit polyclonal anti-TTF-1 or Santa Cruz control

9

rabbit polyclonal IgG) was added to 25 µl of beads and incubated rocking at 4°C for at

least one hour. Antibody-bound beads were washed three times in bead blocking buffer,

resuspended in 100 µl of bead blocking buffer and added to the lysate and IP buffer.

Immunoprecipitations

Antibody-bound beads and cell lysate were incubated in 4.5 ml of IP buffer overnight

rocking at 4°C. Supernatant was saved for immunoblot analysis before washing. Beads

were washed three times in Net IP Wash Buffer (50 mM Tris, pH 8; 150 mM NaCl;

0.05% Triton X-100; Invitrogen Ultra-Pure Water) and then once in Qiagen TE Buffer

before elution in IP Elution Buffer (4% SDS; 0.1 M DTT; 0.1 M Tris-HCL, pH 7.6) at

65°C for 1 hour. Eluate was stored at -20°C before being analyzed by immunoblotting.

Immunoblotting

40 µg of lysate or supernatant was mixed with 10 µl of protein gel loading dye. 35 µl was

loaded into BioRad 4-15% polyacrylamide gels and run at 250 V for approximately 40

minutes. Gel proteins were either transferred onto a PVDF membrane or retained for

Silver Stain analysis. PVDF membrane was blocked with 5% milk-TBST (0.5% Tween-

20) solution and probed with anti-NKX2-1 rabbit polyclonal primary antibody at a

concentration of 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at

4°C. Membrane was washed three times in TBST for 5 minutes at room temperature and

then probed with Protein A-HRP antibody at a concentration of 1:5000 for 1 hour.

Membrane was again washed three times in TBST for 5 minutes at room temperature

before performing autoradiography using chemi-luminescence, using GE ECL reagents.

10

For a positive control in immunoblots using FOXA1, membranes were probed with

FOXA1 primary antibody (Anti-FOXA1 antibody - ChIP Grade (Abcam ab5089)) at 0.5

µg/ml overnight at 4°C, followed by three washes in TBST for 5 minutes at room

temperature. Membranes were then probed with Protein A-HRP antibody at a

concentration of 1:5000 for 1 hour and then followed by three washes in TBST for 5

minutes at room temperature before performing autoradiography using chemi-

luminescence, using GE ECL reagents.

Silver staining

Exact protocol from (Pierce Silver Stain for Mass Spectrometry, prod# 24600) was

followed (Imai & Mische, 1999). Silver stained gels were dried using a vacuum gel dryer.

Mass Spectrometric analysis

Mass spectrometry of co-IP eluates was done by the Stanford University Mass

Spectrometry (SUMS) core facility, using liquid chromatography mass spectrometry

(LC-MS/MS on a Thermo LTQ-Orbitrap Velos instrument) to separate proteins

(trypsinized peptide preparations) on the basis of mass and charge (Imai & Mische,

1999). Samples were sent to SUMS as 25µL of eluate in IP Elution Buffer. Peptides were

mapped to annotated proteins using SEQUEST or Mascot software, and data visualized

using Scaffold (Proteome Software). Candidate binding partners of interest were those

identified in co-IPs by the two different anti-NKX2-1 antibodies (with distinct NKX2-1

epitopes), but not by IgG control. Non-specific proteins, including cytokeratin proteins

presumed to be contaminants, were excluded from consideration.

11

Results

Defining NKX2-1 isoform expression patterns in lung cancer

To investigate which isoform(s) of NKX2-1 are expressed in lung cancer, we performed

an immunoblot

analysis of NKX2-1

using two different

lung cancer cell

lines: HCC1195 and

HCC1833. For

comparison, lysates

from 293T

embryonic kidney

cells, which do not

express endogenous

NXK2-1, were

transfected with

either the NKX2-1

long or short isoform,

and immunoblot

analysis was done running the transfected cell lysates alongside the lung cancer cell

lysates. HCC1195 and HCC1833 were chosen for these experiments because of their

known NKX2-1 amplification and over-expression (Kwei et al., 2008). It has been

previously known that normal lung cells express the short NKX2-1 isoform (Boggaram,

Figure 1. Evaluating NKX2-1 isoform expression in lung cancer cells. A, The two protein isoforms of NKX2-1 differ in size and contain four distinct domains: the N-terminal activation domain (AD), the homeodomain (HD), the inhibitor domain (ID), and the C-terminal activation domain (Boggaram, 2009). B, Immunoblot analysis of NKX2-1 expression in HCC1195 and HCC1833 cells, as well as control 293T cells transfected with either the long (293T Long) or short (293T Short) isoform. Lysates from lung cancer cells and 293T cells were run in parallel to assess the isoform size.

N C

371 aa

401 aa

N-terminal AD

HD ID C-terminalAD

50

37

kD

Long isoform

Short isoform

HCC 1195

HCC 1833

293T Long

293T Short

A

B

12

2009; Yatabe, Mitsudomi, & Takahashi, 2002); however, the isoform(s) expressed in

lung cancer cells were not known. By using the transfected 293T lysates expressing either

the long or short isoform with sizes of approximately 44 kD and 42 kD respectively, the

immunoblot analysis revealed that the short NKX2-1 isoform is the isoform

predominantly expressed in HCC1195 and HCC1833 cell lines.

Evaluating the oncogenic potential of NKX2-1 isoforms in lung cancer

Having identified that the NKX2-1 short isoform is the isoform predominantly expressed

in the two lung cancer cell lines, we next sought to investigate whether the long and short

isoforms have different oncogenic capacity. Our strategy was to knock down endogenous

NKX2-1 expression (by RNA interference) in HCC1833 lung cancer cells, which reduces

cell proliferation, and then determine whether exogenous expression of NKX2-1 long

isoform, short isoform, or both, can “rescue” NKX2-1 knockdown by restoring cell

proliferation. To this effect, we have successfully cloned the long and short NKX2-1

isoforms into lentiviral expression constructs, and have created stable HCC1833 cell lines

expressing either exogenous long or short NKX2-1 isoforms. The decisive experiment

(knocking down endogenous NKX2-1) will be initiated in future work.

Purifying NKX2-1 binding partners by co-immunoprecipitation

Our leading hypothesis is that NKX2-1 associates with distinct protein partners in lung

cancer, which leads to an altered, oncogenic transciptional response. In order to identify

the protein partners NKX2-1 associates with in lung cancer cells, we performed co-

immunoprecipitation experiments, as diagrammed in Figure 2B. Since NKX2-1 is a

13

nuclear transcription factor, we enriched for NKX2-1 by using nuclear lysates. We used

anti-NKX2-1 antibody-bound Protein A magnetic beads to immunoprecipitate NKX2-1.

The immunoprecipitated samples were then subjected to immunoblotting with an NKX2-

1 specific antibody, to verify that NKX2-1 was pulled down in the immunoprecipitation

(Figure 2A). In order to ensure that the co-immunoprecipitation conditions were

favorable to preserving protein interactions, we then proceeded to probe the immunoblot

for FOXA1, a known binding partner of NKX2-1 (Minoo et al., 2007). FOXA1 was used

as a positive control, and its presence in the immunoprecipitated fraction of NKX2-1

confirmed that endogenous protein-

protein interactions were being

preserved (Figure 2A).

50

37

IgG heavy chain

NKX2-1

IgG heavy chain

kDInputNKX2-1 IP

FOXA150

37

A

B

Figure 2. Co-immunoprecipitation of NKX2-1 and known binding partner. A, Immunoblots of input and co-immunoprecipitated protein in HCC1195 show immunoprecipitation of NKX2-1 and co-immunoprecipitation of known binding partner FOXA1. IP was conducted using Santa Cruz anti-NKX2-1 antibody, and Input was loaded at 10% of IP fraction. B, The schematic outlines the steps taken to identify protein binding partners of NKX2-1.

14

Mass spectrometry analysis of co-immunoprecipated proteins

In order to identify

NKX2-1’s

unknown binding

partners in lung

cancer cells, co-

immunoprecipitated

samples were

subjected to mass

spectrometry analysis. Figure 3 shows the immunoblot analysis of the samples submitted

for mass spectrometry analysis, which was done to verify that the co-

immunoprecipitation conditions preserve NKX2-1 but are stringent enough to prevent

Figure 3. Mass spectrometry analysis of NKX2-1 co-immunoprecipitates. Immunoblot of input and NKX2-1 co-immunoprecipitated HCC1195 samples. Input was loaded at 10% of IP fractions. IPs are labeled by the antibody used. Antibodies used for co-immunoprecipitation were Santa Cruz anti-NKX2-1 (SC IP), Seven Hills anti-NKX2-1 (SH IP), and Santa Cruz control rabbit IgG (ctrl IgG IP) for lanes 2, 3 and 4, respectively.

kD

50

37

NKX2-1

InputNKX2-1 SC IP

NKX2-1 SH IP

ctrl IgG IP

Table 1. Mass spectrometry analysis to identify potential protein binding partners of NKX2-1. Table shows seven of the 379 proteins identified in the mass spectrometry analysis. Proteins were included in table on the basis of greater abundance in the NKX2-1 samples IP’d with Santa Cruz (SC IP) and Seven Hills (SH IP) antibodies, and lower abundance in the control IgG sample (Ctrl IgG IP). Proteins were excluded on the basis of determined non-specific binding or contamination from serum or other sources (e.g. cytokeratins). Proteins were organized in the table in decreasing order of peptide counts for SC and SH IP fractions.

15

non-specific binding, as evidenced by the absence of protein in the control IgG IP lane.

Analysis from mass spectrometry identified a total of 379 proteins found in the submitted

samples. Of those 379 proteins, a select number were considered to be candidate NKX2-

1-assocatied proteins (Table 1), based upon elevated levels of these proteins in the

immunoprecipitations done with the two different anti-NKX2-1 antibodies (Santa Cruz

and Seven Hills), as compared to the control IgG IP fraction.

Optimization of co-immunoprecipitation conditions

In light of the relatively large number of proteins that were identified as candidate

binding partners in the first mass spectrometry analysis, we sought to further optimize the

Figure 4. Comparison of two co-immunoprecipitation protocols and conditions to eliminate non-specific binding. A, The standard IP protocol was followed in HCC1195 cells, with the removal of 1% SDS, to reduce stringency. B, Co-immunoprecipitation was done in HCC1833 cells using the Active Motif Universal Co-IP Kit for nuclear isolation of protein and then subsequent steps from standard IP protocol. For both sets of IPs, IPs are labeled by antibody used, which were Santa Cruz anti-NKX2-1 (SC IP), Seven Hills anti-NKX2-1 (SH IP), and Santa Cruz control rabbit IgG (ctrl IgG IP) for lanes 2, 3 and 4, respectively. Immunoblots were probed separately with anti-NKX2-1 (Santa Cruz) and anti-FOXA1 (Abcam) antibodies (a known binding partner of NKX2-1).

50

37

50

37

InputNKX2-1 SC IP

NKX2-1 SH IP

ctrl IgG IP

kD

NKX2-1

NKX2-1

50

50

FOXA1

FOXA1

Standard co-IP protocol,without SDS

Hybrid co-IP protocol: Active Motif Kit nuclear lysate prep and standard IP

A

B

16

co-immunoprecipitation methods to obtain greater specificity in binding partners. To do

so, a comparison was done between two co-immunoprecipitation methods—the co-IP

protocol previously used (Figure 2A, 3), and a hybrid protocol of Active Motif Universal

Co-IP Kit and the standard co-IP protocol, because we found through repeated

experiments and mass spectrometry results that conditions were not optimal for

preserving potential interactions while eliminating non-specific ones. When we used the

hybrid protocol of the Active Motif kit for lysate preparation, which does not use Sodium

dodecyl sulfate (SDS) in the nuclei lysis preparation (which may have been one of the

causes of inefficient immunoprecipitation), and subsequent immunoprecipitation with the

standard IP protocol to perform a co-IP, the non-specific binding of NKX2-1 to control

IgG disappeared.

Cross-linking proteins to preserve protein interactions with NKX2-1

In order to further ensure that protein-protein interactions were being preserved during

the co-immunoprecipitation steps, proteins were cross-linked by first treating the cells

with a chemical cross-linking agent, Dithiobis[succinimidyl propionate] (DSP) (Zlatic et

al., 2010). Following cross-linking, lysate preparation and immunoprecipitation were

done, after which cross-links were either retained or reversed (using dithiothreitol; DTT),

and then proteins were examined by immunoblot analysis. As predicted, NKX2-1 is

visible as a single band in the reversed fractions, whereas, there is a higher-molecular

weight smear of NKX2-1-bound proteins in the cross-linked fractions (Figure 5).

Identification of the cross-linked NKX2-1 bound protein by mass spectrometry is now

underway.

17

50

37

75

100

150

250

NKX2-1

NKX2-1 protein binding complexes

kD+ + + +- - - -Input IP - SC IP - SH IP - IgG

Cross-links

Figure 5. Cross-linking proteins in cultured cells and subsequent immunoprecipitation. HCC1833 cells were cross-linked, followed by lysate preparation (Active Motif protocol) and immunoprecipitation (standard IP protocol). Cross-links were then reversed using 0.1 M DTT before immunoblot analysis was conducted. As in previous experiments, antibodies used for IP were Santa Cruz anti-NKX2-1, Seven Hills anti-NKX2-1, and Santa Cruz control rabbit IgG for lanes 2, 3 and 4, respectively. Immunoblots was done probing with the Santa Cruz anti-NKX2-1 antibody.

18

Discussion/Conclusions

The goals of this project were: 1) to determine which isoforms of NKX2-1 are

expressed in certain lung cancer cells, and characterize their oncogenic capacity; and 2)

to identify cancer-relevant protein binding partners of NKX2-1 in lung cancer cells.

Although it has been previously shown that NKX2-1 is amplified and overexpressed in

some lung cancer cell lines (Kwei et al., 2008), the isoforms and potential protein binding

partners involved in its oncogenicity have not been well explored. Whereas some

characteristics have been identified for NKX2-1 in normal lung cells, it is unclear how

many of those characteristics are shared in lung cancer cells with amplified and

overexpressed NKX2-1, and whether those differences might underlie NKX2-1’s

contribution to cancer.

Our study provides the first insight that the short isoform of NKX2-1 is the

isoform predominantly expressed in lung cancer, although evaluating additional cell lines

is needed to confirm our findings. It has been previously reported that NKX2-1 has two

isoforms, and that the short isoform is known to be expressed in certain normal human

lung cell types, while expression of the long isoforms in human lung cells is still not clear

(Boggaram, 2009; Kolla et al., 2007). Using immunoblot analysis of NKX2-1-transfected

293T cells compared to endogenously expressing lung cancer cell lines HCC1195 and

HCC1833, we determined that the lung cancer cells predominantly express the short

isoform. Ongoing experiments are looking at the effect of the isoform (long versus short)

on oncogenic potential, using exogenous isoforms introduced into HCC1833 cells, where

the endogenous NKX2-1 can be selectively depleted by RNA interference. This should

19

provide novel insight into the role and relative importance of these two isoforms in lung

cancer cells.

In lung cancer cells, as yet little is known or understood about the proteins with

which NKX2-1 interacts. One known binding partner is transcription factor FOXA1;

however, there are likely several other proteins that may be important in determining

where on the genome NKX2-1 localizes and how it behaves. Our study hypothesizes that

NKX2-1’s oncogenic activity stems at least in part from its protein binding partners, and

we use co-immunoprecipitation and subsequent mass spectrometry analysis to identify

potential protein binding partners. An initial survey of NKX2-1 associated proteins by

mass spectrometry identified the presence of SMARCA5, HNRNPCL1, HMGA2,

BAZ2A, and HIST1H1A, some of which may be interesting targets for further validation

and experimentation. These proteins are of particular interest given their known

regulatory functions in the cell, which could explain their direct association with NKX2-

1. Notably, SMARCA5, which is understood to modulate gene expression, DNA repair

and replication, and chromatin structure, has been implicated in multiple types of cancers,

including prostate, breast and gastric cancers (Chen et al., 2014; Reis et al., 2013; Leite et

al., 2013). Therefore, it may not be surprising if further experiments reveal a role in lung

cancer. Also of significant interest is further elucidating the role of HMGA2 in lung

cancer, as research has already shown that NKX2-1 and HMGA2 are involved in the

same pathway in lung cancer proliferation, via microRNA activity (Kumar et al., 2014;

Xiang, Q et al., 2013; Rice et al., 2013). Given that HMGA2 is already known to interact

with NKX2-1 in some way, we would like to validate these prior findings by determining

their direct protein interactions. We will begin to verify these interactions by probing the

20

NKX2-1 co-immunoprecipitation blots for the presence of these other proteins, and then

also doing the reverse experiment by co-immunoprecipitating the potential binding

partner (e.g. SMARCA5) and subsequently probing for NKX2-1. For further analysis,

particularly for the potential binding partners whose ties to NKX2-1 have not previously

been explored, we would conduct a comparison between normal and lung cancer cells to

determine if these interactions are unique to lung cancer cells. Ideally, these preliminary

validations of interaction with NKX2-1 will lead to further analysis of the roles these

potential binding partners play in driving NKX2-1’s oncogenicity.

Several questions remain after this analysis, including how best to optimize co-

immunoprecipitation conditions to preserve relevant protein-protein interactions while

eliminating non-specific ones. Multiple iterations revealed that a combination of co-

immunoprecipitation protocols yielded the best results, although some non-specific

binding was still detected in the control IgG IP fraction. Subsequent experiments have

tried to preserve protein interactions by cross-linking before performing co-

immunoprecipitation. Although the mass spectrometry results from cross-linking are

pending, immunoblots revealed that there is a difference in protein detection and pattern

between cross-linked and reversed fractions, suggesting that NKX2-1 protein binding

complexes are present and should be identifiable by mass spectrometry in ongoing and

future experiments. By comparing future mass spectrometry data to the existing analysis,

we can also assess how consistent our co-immunoprecipitations are, because we should

see the same significant proteins over multiple iterations. To validate the proteins

identified by mass spectrometry, we will follow up with comparisons between normal

21

lung and lung cancer cells, and ultimately with siRNA-mediated knockdown experiments

of potential protein partners, to assess their necessity for lung cancer growth.

In conclusion, we identified that the short isoform of NKX2-1 is the isoform

predominantly expressed in lung cancer cells; optimized co-immunoprecipitation

conditions for pulling down NKX2-1 and potential binding partners; and determined that

there are proteins and protein complexes binding to NKX2-1 in lung cancer cells, which

have set the stage for further analysis of NKX2-1 in lung cancer cells to identify and

describe oncogenic characteristics and co-conspirators of this lung cancer oncogene.

22

Bibliography Boggaram, V. (2009). Thyroid transcription factor-1 (TTF-1/Nkx2.1/TITF1) gene

regulation in the lung. Clinical Science (London, England  : 1979), 116(1), 27–35. Cao, Y., Vo, T., Millien, G., Tagne, J. B., Kotton, D., Mason, R. J., Williams, M. C., and

Ramirez, M. I. (2010) Epigenetic mechanisms modulate thyroid transcription factor 1-mediated transcription of the surfactant protein B gene. J. Biol. Chem. 285, 2152–2164.

Chen, D., Sun, Y., Yuan, Y., Han, Z., Zhang, P., Zhang, J., … Ma, L. (2014). miR-100 induces epithelial-mesenchymal transition but suppresses tumorigenesis, migration and invasion. PLoS Genetics, 10(2), e1004177.

Das, A., Acharya, S., Gottipati, K. R., Mcknight, J. B., Chandru, H., Alcorn, J. L., & Boggaram, V. (2011). Thyroid transcription factor-1 ( TTF-1 ) gene  : identification of ZBP-89 , Sp1 , and TTF-1 sites in the promoter and regulation by TNF-alpha in lung epithelial cells, 1(28).

Ikeda, K., Clark, J., & Shaw-White, J. (1995). Gene Structure and Expression of Human Thyroid Transcription Factor-1 in Respiratory Epithelial Cells. Journal of Biological Chemistry.

Imai, B., & Mische, S. (1999). mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis.

Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T., & Thun, M. J. (2008). Cancer statistics, 2008. CA: A Cancer Journal for Clinicians, 58(2), 71–96. doi:10.3322/CA.2007.0010

Kendall J, Liu Q, Bakleh A, Krasnitz A, Nguyen KC, Lakshmi B et al. (2007). Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer. Proc Natl Acad Sci USA 104: 16663–16668.

Kimura, S., Hara, Y., Pineau, T., Fernandez-Salguero, P., Fox, C. H., Ward, J. M., & Gonzalez, F. J. (1996). The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes & Development, 10(1), 60–69.

Kolla, V., Gonzales, L. W., Gonzales, J., Wang, P., Angampalli, S., Feinstein, S. I., & Ballard, P. L. (2007). Thyroid transcription factor in differentiating type II cells: regulation, isoforms, and target genes. American Journal of Respiratory Cell and Molecular Biology, 36(2), 213–25.

Kumar, M. S., Armenteros-Monterroso, E., East, P., Chakravorty, P., Matthews, N., Winslow, M. M., & Downward, J. (2014). HMGA2 functions as a competing endogenous RNA to promote lung cancer progression. Nature, 505(7482), 212–7.

Kwei, K. a, Kim, Y. H., Girard, L., Kao, J., Pacyna-Gengelbach, M., Salari, K., … Pollack, J. R. (2008). Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene, 27(25), 3635–40.

Leite, K. R. M., Morais, D. R., Reis, S. T., Viana, N., Moura, C., Florez, M. G., … Srougi, M. (2013). MicroRNA 100: a context dependent miRNA in prostate cancer. Clinics (São Paulo, Brazil), 68(6), 797–802.

23

Minoo, P., Hu, L., Xing, Y., Zhu, N. L., Chen, H., Li, M., … Li, C. (2007). Physical and functional interactions between homeodomain NKX2.1 and winged helix/forkhead FOXA1 in lung epithelial cells. Molecular and Cellular Biology, 27(6), 2155–65.

Minoo P, Su G, Drum H, Bringas P, Kimura S. (1999). Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(-/-) mouse embryos. Dev Biol 209: 60–71.

Non-Small Cell Lung Cancer Treatment (PDQ®) - National Cancer Institute. (n.d.). Ramirez, M. I., Rishi, A. K., Cao, Y. X., and Williams, M. C. (1997) TGT3,

thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of T1, a lung alveolar type I cell gene. J. Biol. Chem. 272, 26285–26294.

Reis, S. T., Timoszczuk, L. S., Pontes-Junior, J., Viana, N., Silva, I. a, Dip, N., … Leite, K. R. M. (2013). The role of micro RNAs let7c, 100 and 218 expression and their target RAS, C-MYC, BUB1, RB, SMARCA5, LAMB3 and Ki-67 in prostate cancer. Clinics (São Paulo, Brazil), 68(5), 652–7.

Rice, S. J., Lai, S.-C., Wood, L. W., Helsley, K. R., Runkle, E. A., Winslow, M. M., & Mu, D. (2013). MicroRNA-33a mediates the regulation of high mobility group AT- hook 2 gene (HMGA2) by thyroid transcription factor 1 (TTF-1/NKX2-1). The Journal of Biological Chemistry, 288(23), 16348–60.

Salari, K., Spulak, M. E., Cuff, J., Forster, A. D., Giacomini, C. P., Huang, S., … Pollack, J. R. (2012). CDX2 is an amplified lineage-survival oncogene in colorectal cancer. Proceedings of the National Academy of Sciences of the United States of America, 109(46), E3196–205.

Shaw-White, J. R., Bruno, M. D., and Whitsett, J. A. (1999) GATA-6 activates transcription of thyroid transcription factor-1. J. Biol. Chem. 274, 2658 –2664.

Society, A. C. (2008). Cancer facts & figures. Tanaka, H., Yanagisawa, K., Shinjo, K., Taguchi, A., Maeno, K., Tomida, S., …

Takahashi, T. (2007). Lineage-specific dependency of lung adenocarcinomas on the lung development regulator TTF-1. Cancer Research, 67(13), 6007–11.

Tian J, Mahmood R, Hnasko R, Locker J. (2006). Loss of Nkx2.8 deregulates progenitor cells in the large airways and leads to dysplasia. Cancer Res 66: 10399–10407.

Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC. (2004). Pathology & Genetics: Tumours of the lung, pleura, thymus and heart. (World Health Organization Classification of Tumours). IARC Press: Lyon.

Varma, S., Cao, Y., Tagne, J.-B., Lakshminarayanan, M., Li, J., Friedman, T. B., … Ramirez, M. I. (2012). The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation. The Journal of Biological Chemistry, 287(44), 37282–95.

Yatabe, Y., Mitsudomi, T., & Takahashi, T. (2002). TTF-1 Expression in Pulmonary Adenocarcinomas. The American Journal of Biological Chemistry.

Xiang Q, Tang H, Yu J, Yin J, Yang X, Lei X. (2013). MicroRNA-98 sensitizes cisplatin-resistant human lung adenocarcinoma cells by up-regulation of HMGA2. Pharmazie, 68:274–81.

Zlatic, S. a, Ryder, P. V, Salazar, G., & Faundez, V. (2010). Isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography. Journal of Visualized Experiments  : JoVE, (37), 5–8.