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bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.04.433941
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this version posted March 4, 2021. The copyright holder for this preprint
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It is made
available under a
CC-BY-NC-ND 4.0 International license . Introduction
Lung cancer and specifically lung adenocarcinoma (LUAD) remains the leading cause of
cancer death world-wide. New therapies that molecularly target driver mutations have improved
patient outcome. However, the development of resistance mut... |
In KRASG12D-driven tumors, NKX2-1 deletion increases phospho-ERK
and accelerates progression to invasive carcinoma and metastasis (8, 24, 27). Clinically, low
NKX2-1 expression portends a poor prognosis (28-30). We reasoned that in addition to beginning
the de-differentiation process, a repressed-NKX2-1 status might dr... |
We found that NKX2-1 loss in LUAD clinical samples and cell lines correlates with reduced
DUSP6. Further, NKX2-1 directly induces DUSP6 expression and limits tumor cell proliferation,
migration, invasion, and tumor growth metastasis. While DUSP6 knockout can be toxic, it
abrogates the effects of NKX2-1 on cell prolifer... |
1D). NKX2-1 did not induce
SPRY2 protein in any cell line (A549 p=0.617, H1299 p=0.098, H23 p=0.493, Fig. 1D). We also tested if NKX2-1 induces DUSP6 or SPRY2 in a murine LUAD cell line derived
from KRASG12D; TP53Null; NKX2-1Null mouse tumors (3658 cells, (24)). Indeed, qRT-PCR again
showed that exogenous NKX2-1 increa... |
The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under a
CC-BY-NC-ND 4.0 International license . lines, including A549 and 3658 cells (50). However, A549 and 3658 cells are ... |
In our LUAD cells with re-
introduction of NKX2-1, we found that the p-ERK induced by stimulation with RAS pathway
agonists EGF or PMA trended lower in cells expressing NKX2-1 compared to cells with empty
vector (Fig. S2C, S2D, not significant). The small change is consistent with the previous studies
on DUSP6. NKX2-1 ... |
5 out of 9 mice with A549-V tumors exhibited
micrometastases versus 0 out of 9 mice with A549-NKX2-1 tumors (Fig. 3B). IHC showed that
tumors lacking NKX2-1 exhibit low DUSP6 expression and high p-ERK (Fig. 3C). In contrast,
tumors with NKX2-1 reconstitution exhibited high DUSP6 expression and reduced p-ERK (Fig. 3C). ... |
S4B). In H1299 cells, DUSP6 loss did not change cell
proliferation (Day 6 p=0.38), but SPRY2 loss increased proliferation (Day 5 p=7.5x10-6, Fig. S4D). This suggests that in cell culture, A549 and H1299 cells engage compensatory signaling to limit
the effects of DUSP6 loss. However, under these unpressured growth condi... |
To test
this, we generated subcutaneous tumors with the A549 DUSP6 knockouts harboring inducible
TRE-NKX2-1 and GFP-luciferase. After the tumors reached 150-400 mm3 (5 weeks for control
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.04.433941
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this version posted March 4, 2021. The copyright holder for this pr... |
It is made
available under a
CC-BY-NC-ND 4.0 International license . expression reduced tumor size and p-ERK in this system (Fig. 5F). In summary, DUSP6 and
SPRY2 are sufficient to limit tumorigenesis in the genetically engineered model of LUAD. Discussion
Activating mutations in EGFR, KRAS and BRAF initiate LUAD tumor... |
Further, DUSP6
but not SPRY2, protein was significantly upregulated upon introduction of NKX2-1 into human
LUAD cell lines, both in vitro and in xenografts. However, in the genetically engineered mouse
tumors, Nkx2-1 deletion caused a loss of SPRY2 along with a loss of DUSP6. This suggests that
NKX2-1 works through DUS... |
Similarly, LUAD cells with
acquired resistance to EGFR inhibitors are addicted to the inhibitor-dampened ERK activity such
that inhibitor withdrawal induces toxic ERK hyperactivation (65). Lineage heterogeneity promotes
chemoresistance (26). Models of tumors with heterogeneous ERK activity show that ERK
heterogeneity c... |
A. Proliferation of A549 and H1299 cells. Significance from one-way ANOVA. B. and C.
Random-walk migration velocity and directionality of H1299 cells upon NKX2-1 expression and
MEK inhibition (Selumetinib 10 µM). Velocity is Mean Squared Displacement. Significance from
bioRxiv preprint
doi:
https://doi.org/10.1101/202... |
Figure S3. NKX2-1 is required for DUSP6 expression and ERK activity in vivo. A. and B. Representative IHC images of KRASG12D;NKX2-1WT and KRASG12D;NKX2-1Null tumors:
NKX2-1 target pro-SPB, DUSP6, SPRY2, p-ERK and effector p-RSK and p-S6 levels. Scale bars:
100 µm. n=6 mice tamoxifen, n=6 mice corn oil injections. Figur... |
Relative expression compared to total ERK. Means and
SD from n=3 independent experiments. C. IHC for DUSP6 in TRE- Spry2 tumors and SPRY2 in
TRE-DUSP6 tumors. Methods
Histology and IHC
Human tissue microarrays were formalin fixed and paraffin embedded: US Biomax
BCS04017, BCS04017a, LC10014a. Arrays were stained for NK... |
The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under a
CC-BY-NC-ND 4.0 International license . with AscI and EcoRI, and ligated into a modified pBabe-Puro retroviral backb... |
2011 PMID 21458673
MGI 4950589
Dr. Scott Lowe (MSK, New York, New York)
Cell line
293T
ATCC
# CRL-11268
Cell line Cell line
Phoenix-Ampho A549
ATCC ATCC
# CRL-3213 # CCL-185
Cell line
H1299
ATCC
# CRL-5803
Cell line Cell line
Virus
Plasmid
Plasmid
H23 3658
Ad5CMV-FlpO
MSCV-NKX2-1
pGL3-191p pGL3-508p
ATCC Snyder et al. ... |
Cells were tested for mycoplasma every 3-4
bioRxiv preprint
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Cells were selected with
blasticidin for 1 week. NKX2-1 expression was induced in cells with 1 µg/ml of doxycycline and
confirmed by Western blotting. Ultracentrifugation at 25,000 rpm for 2 hrs was necessary to
concentrate pCDH-TRE-DUSP6 and pCDH-TRE-SPRY2 virus for in vivo infection. Fluorescence-activated cell sorti... |
Cells were transfected the next day with pRenilla (Addgene) and empty pGL3 vector
or pGL3 vector containg the DUSP6 promoter regions 191p and 508p using Lipofectamine 2000
(Invitrogen). After 24 hours the Dual-Glo Luciferase Assay System (Promega) was used and
luminescence was measured by the Synergy HT microplate read... |
Spheroids
bioRxiv preprint
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CC-B... |
Genetically engineered mice and tumor initiation
Under University of Utah IACUC #18-08005, mice harboring KRasfrtSfrt-G12D/+;Nkx2-1f/f;
RosafrtSfrt-CreERT2 alleles were infected intratracheally with 1 x 107 pfu/mouse Ad5CMV-FlpO
(University of Iowa Viral Vector Core). FlpO recombined Frt-STOP-Frt for simultaneous KRASG... |
Nature. 2014;511(7511):543-50. doi: 10.1038/nature13385. PubMed PMID:
25079552. 3. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, Sougnez C, Greulich H,
Muzny DM, Morgan MB, Fulton L, Fulton RS, Zhang Q, Wendl MC, Lawrence MS, Larson DE,
Chen K, Dooling DJ, Sabo A, Hawes AC, Shen H, Jhangiani SN, Lewi... |
8. Jackson EL, Olive KP, Tuveson DA, Bronson R, Crowley D, Brown M, Jacks T. The
differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer research. 2005;65(22):10280-8. doi: 10.1158/0008-5472.CAN-05-2193. PubMed PMID: 16288016. 9. Noguchi M. Stepwise progression of pulmonary adenocarcinoma--cli... |
Epub 2017/08/08. doi:
10.1038/nature23297. PubMed PMID: 28783725; PMCID: PMC5648056. 17. Cicchini M, Buza EL, Sagal KM, Gudiel AA, Durham AC, Feldser DM. Context-Dependent
Effects of Amplified MAPK Signaling during Lung Adenocarcinoma Initiation and Progression. Cell reports. 2017;18(8):1958-69. Epub 2017/02/24. doi: 1... |
doi: 10.1038/modpathol.2010.232. PubMed PMID: 21252858. 24. Snyder EL, Watanabe H, Magendantz M, Hoersch S, Chen TA, Wang DG, Crowley D,
Whittaker CA, Meyerson M, Kimura S, Jacks T. Nkx2-1 represses a latent gastric differentiation
program in lung adenocarcinoma. Molecular cell. 2013;50(2):185-99. doi:
10.1016/j.molcel... |
2015;21(15):3480-91. Epub 2015/04/17. doi: 10.1158/1078-0432.CCR-14-
3286. PubMed PMID: 25878335; PMCID: PMC4526428. 31. Gillies TE, Pargett M, Silva JM, Teragawa C, McCormick F, Albeck JG. Oncogenic mutant
RAS signaling activity is rescaled by the ERK/MAPK pathway. bioRxiv. 2020:2020.02.17.952093. doi: 10.1101/2020.02... |
and clinical outcome in non-small-cell lung cancer. N Engl J Med. 2007;356(1):11-20. Epub
2007/01/05. doi: 10.1056/NEJMoa060096. PubMed PMID: 17202451. 40. Okudela K, Yazawa T, Woo T, Sakaeda M, Ishii J, Mitsui H, Shimoyamada H, Sato H, Tajiri
M, Ogawa N, Masuda M, Takahashi T, Sugimura H, Kitamura H. Down-regulation o... |
2013;27(2):197-210. Epub 2013/01/17. doi: 10.1101/gad.203208.112. PubMed PMID:
23322301; PMCID: 3566312. 47. Ding W, Bellusci S, Shi W, Warburton D. Functional analysis of the human Sprouty2 gene
promoter. Gene. 2003;322:175-85. Epub 2003/12/04. doi: 10.1016/j.gene.2003.09.004. PubMed
PMID: 14644509. 48. Ekerot M, Stav... |
Genes & development. 1996;10(1):60-9. Epub
1996/01/01. doi: 10.1101/gad.10.1.60. PubMed PMID: 8557195. 56. Sutherland KD, Song JY, Kwon MC, Proost N, Zevenhoven J, Berns A. Multiple cells-of-
origin of mutant K-Ras-induced mouse lung adenocarcinoma. Proceedings of the National
Academy of Sciences of the United States o... |
It is made
available under a
CC-BY-NC-ND 4.0 International license . 2019;25(13):4117-27. Epub 2019/04/03. doi: 10.1158/1078-0432.CCR-18-3224. PubMed PMID:
30936125; PMCID: PMC6606396. 63. Shojaee S, Caeser R, Buchner M, Park E, Swaminathan S, Hurtz C, Geng H, Chan LN,
Klemm L, Hofmann WK, Qiu YH, Zhang N, Coombes KR, ... |
Image-guided genomics of
phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective
cancer invasion. Nat Commun. 2017;8:15078. Epub 2017/05/13. doi: 10.1038/ncomms15078. PubMed PMID: 28497793; PMCID: PMC5437311. bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.04.433941
;
this... |
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.04.433941
;
this version posted March 4, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under a
CC-BY-NC-ND 4.0... |
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.30.437768
;
this version posted March 30, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Caspase-2 regulates S-phase cell cycle events to protect
f... |
Thus, our data supports a model where caspase-2 regulates cell cycle
events to protect from the accumulation of DNA damage independently of its pro-apoptotic
function. 4
bioRxiv preprint
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https://doi.org/10.1101/2021.03.30.437768
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this version posted March 30, 2021. The copyright holder for this preprint
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This has been noted for
caspase-2 dependent apoptosis that can be either p53-dependent12-14 or p53-independent.15, 16
Caspase-2 has been shown to be activated by several different inducers of DNA damage
including etoposide, cisplatin, and camptothecin.13, 17-19 However, apoptosis can often proceed
in the absence of cas... |
Mouse embryonic
fibroblasts (MEF) were grown in the same medium supplemented with sodium pyruvate (1 mM),
1X non-essential amino acids, and beta-mercaptoethanol (55 µM). Litter-matched Casp2+/+ and
Casp2-/- MEF were generated and transduced with E1A and Ras as previously described.19
Briefly, early passage MEF were sim... |
Cells were imaged using one of two microscope systems. The first is a spinning disk confocal
microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA), equipped with a CSU-X1A 5000
spinning disk unit (Yokogawa Electric Corporation, Japan), multi laser module with wavelengths
of 458 nm, 488 nm, 514 nm, and 561 nm, and an... |
Images were smoothed by Gaussian
method followed by separation using Otsu with an adaptive thresholding strategy. Cells were
then declumped by shape and automatically counted. For quantitation of
γ
H2AX foci, the
number of foci per cell was calculated from RGB TIFF images using FoCo, a graphical user
interface that use... |
Cells
were washed in BD Perm/Wash buffer and centrifuged at 300 x g between each step. To
uncover the BrdU epitope, the cells were treated with DNAse (30 µg per 106 cells) for 1 h at
37°C, washed in BD Perm/Wash buffer, and centrifuged at 300 x g. The cells were then
incubated in a 1:50 dilution of FITC-labeled anti-Br... |
ImageJ software was used to analyze the DNA fibers. For each data set,
about 300 fibers were counted for stalled forks, new origins, or elongated forks. Viability assay
Casp2+/+ and Casp2-/- MEF were plated in a 24-well plate at the indicated densities. After four
days colonies were visualized by staining overnight wit... |
To
confirm these results, we used BrdU/7AAD staining to determine the cell cycle profiles of cycling
Casp2+/+ and Casp2-/- MEF and found that Casp2-/- cells had significantly more cells in S-phase
and less cells in G1-phase compared to the Casp2+/+ cells (Figure 1D, E). The increased
proportion of S-phase cells suggest... |
This indicates that caspase-2 activation does not
coincide G2/M phase of the cell cycle, but is activated in G1 and early S-phase. Loss of caspase-2 results in S-phase specific chromosomal aberrations and delayed exit
16
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.30.437768
;
this version posted March 30, 20... |
HU induces
replication fork stalling and S-phase arrest by depleting the available nucleotide pool for DNA
polymerases.34 Cells were pulse-labeled with 5-iododeoxyuridine (IdU), treated with HU for 2 h
to induce replicative stress, and then washed and pulse-labeled with 5-chlorodeoxyuridine
(CldU). Individual DNA fiber... |
We measured
checkpoint activation in Casp2+/+ and Casp2–/– MEF treated with HU (Figure 5A). Chk1 was
phosphorylated immediately after HU release to a similar extent with and without caspase-2 and
minimal differences in Chk1 phosphorylation between the two cell lines were noted over time. Interestingly, phosphorylation ... |
ATM phosphorylation was not induced by HU in the U2OS cells. In
HeLa cells and in U2OS cells, we detected strong Chk2 phosphorylation in response to HU and
20
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.30.437768
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this version posted March 30, 2021. The copyright holder for this preprint
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22
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.30.437768
;
this version posted March 30, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Discussion
Here we report that caspase-2 is activated ... |
This would suggest that caspase-2 functions downstream of,
or independently of, ATR and Chk1 in response to stalled replication forks. Chk1 has been
proposed as an inhibitor of caspase-2 in response to IR-induced DNA damage.15 Inhibition of
Chk1 potentiates caspase-2 dependent apoptosis by derepressing ATM-mediated
pho... |
Treatment with inducers of G2/M arrest including nocodazaole and Plk1 inhibition has been
shown to induce caspase-2-dependent apoptosis as a mechanism to remove aneuploidy cells.54
However, it is not clear whether caspase-2 regulates cell cycle during the DNA damage
response through activation of the same pathway compo... |
The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. independently of caspase-2 to activate ATR, but the nucleolar PIDDosome is required for
efficient resolution of stalled forks and prevention of DSBs. To fully u... |
*p<0.05. (C) Representative images are shown from the 0 h
and 72 h time points. Nuclei are shown in blue. Bar, 200 µm. (D) Untreated, cycling litter-
matched Casp2+/+ and Casp2-/- E1A/Ras MEF were stained with BrdU/7-AAD and analyzed by
flow cytometry. Representative flow plots are shown. (E) The percentage of cells in... |
The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. three times. **p<0.01. (B) Representative images of metaphase spread of untreated (-) or
irradiated Casp2+/+ and Casp2-/- MEF. Red arrows show breaks; blue arro... |
Bar, 50 µm. 36
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.03.30.437768
;
this version posted March 30, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ed Figure 5. Loss of caspase-2 does not im... |
Results are an average of 3 independent experiments plus or minus
standard deviation. (D) Casp2+/+ and Casp2-/- MEF stably expressing vector or Bcl-XL were
treated with the indicated doses of camptothecin for 4 h or 24 h. Apoptosis was measured at 24
h by Annexin V staining. Results are the average of 3 independent exp... |
J Biol Chem 277: 29803-29809. 14
Baptiste-Okoh N, Barsotti A, Prives C (2008). A role for caspase 2 and PIDD in the process of p53-mediated apoptosis. . Proc Natl Acad Sci U S A 105: 1937-1942. 15
Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R et al (2008). Chk1 suppresses a caspase-2 apoptotic response to... |
Mol Cell Biol 23: 8363-8376. 29
Hunt CR, Dix DJ, Sharma GG, Pandita RK, Gupta A, Funk M et al (2004). Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice. Mol Cell Biol 24: 899-911. 30
Baliga BC, Read SH, Kumar S (2004). The biochemical mechanism of caspase-2 activation. Cell Death ... |
Journal of Biological Chemistry 280: 4649- 4655. 45
Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES (2002). Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 277: 13430-13437. 46
Bonzon C, Bouchier-Hayes L, Pagliari LJ, Green DR, Newmeyer DD (2006). Caspase-... |
ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell 66: 801-817. 60
Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R et al (2008). Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl- 2, and Caspase-3. Cell 133: 864-877. 61
Abraham RT (2001). Ce... |
Nucleic Acids Research 46: 1847-1859. 75
Ando K, Parsons MJ, Shah RB, Charendoff CI, Paris SL, Liu PH et al (2017). NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus. Journal of Cell Biology 216: 1795-1810. 76
Logette E, Schuepbach-Mallepell S, Eckert MJ, Leo XH, Jaccard B, Manzl C et al (2011). PI... |
bioRxiv preprint
doi:
https://doi.org/10.1101/2021.06.30.450599
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this version posted July 1, 2021. The copyright holder for this preprint (which
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Title:
Genome-wide association study and functional validati... |
Barker Foundation, The McCune Foundation
Acknowledgments: The authors would like to acknowledge the neuropathology core of the Massachusetts Alzheimer Disease Research Center, the Biosample Management Repository at Genentech/Roche, the brain repository at UCI, Knight Alzheimer Disease Research Center Neuropathology Cor... |
The copyright holder for this preprint (which
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21
Departments of Pathology (Neuropathology) and Neurological Sciences, Rush University Medical Center, Chicago IL, USA
22
Department of Psychiatry, Alzheimer's... |
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