USP28 enables oncogenic transformation of respiratory cells and its inhibition 1
potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K. 2
3
Cristian Prieto-Garcia
1,2,3
, Oliver Hartmann
1,2
, Michaela Reissland
1,2
, Fabian 4
Braun
1,2
, Süleyman Bozkurt
4
, Carmina Fuss
1,2,5
, Christina Schülein-Völk
6
, Alexander 5
Buchberger
7
, Marco A. Calzado Canale
8,9,10
, Mathias Rosenfeldt
2,11
, Ivan Dikic
3,12
, 6
Christian Münch
4
& Markus E. Diefenbacher
1,2*
7
8
1
Protein Stability and Cancer Group, University of Wuerzburg, Department of Biochemistry and 9
Molecular Biology, Wuerzburg, Germany 10
2
Mildred Scheel Early Career Center, Wuerzburg, Germany 11
3
Molecular Signaling Group, Institute of Biochemistry II, Goethe University Frankfurt, Germany 12
4
Protein quality control, Institute of Biochemistry II, Goethe University Frankfurt, Germany
13
5
Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, 14
University of Wuerzburg, Wuerzburg, Germany 15
6
Core Unit High-Content Microscopy, Biocenter, University of Wuerzburg, Germany 16
7
Department of Biochemistry, Biocenter, University of Wuerzburg, 97074 Würzburg, Germany 17
8
Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain 18
9
Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, 19
Spain 20
10
Hospital Universitario Reina Sofía, Córdoba, Spain 21
11
Institut für Pathologie, Universitaetsklinikum Wuerzburg 22
12
Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Germany. 23
*corresponding author 24
25
26
Keywords: USP28, c-MYC, c-JUN, lung cancer, EGFR, PIK3CA, BRAF, HRAS 27
Gefitinib, Buparlisib, Vemurafenib. 28
29
Financial Support: C.P.G. and O.H. are supported by the German Cancer Aid via 30
grant 70112491. M.R. is funded by the DFG-GRK 2243 and IZKF B335. M.E.D. is 31
funded by the German Israeli Foundation grant 1431. 32
33
*Corresponding Author: Dr. Markus E. Diefenbacher. Lehrstuhl für Biochemie und 34
Molekularbiologie, Biozentrum, Am Hubland, Würzburg, 97074, Germany. Phone: 35
0931 31-88167; Fax: 0931 31-84113; E-mail: markus.diefenbacher@uni-36
wuerzburg.de 37
38
Conflict of Interest: The authors declare no potential conflicts of interest. 39
40
41
42
43
44
45
46
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47
Abstract 48
Oncogenic transformation of lung epithelial cells is a multi-step process, frequently 49
starting with the inactivation of tumor suppressors and subsequent activating 50
mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells 51
undergoing transformation have to adjust to changes, such as metabolic 52
requirements. This is achieved, in part, by modulating the protein abundance of 53
transcription factors, which manifest these adjustments. Here, we report that the 54
deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein 55
abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and
∆
NP63, at 56
early stages of malignant transformation. USP28 is increased in cancer compared to 57
normal cells due to a feed-forward loop, driven by increased amounts of oncogenic 58
transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, 59
interference with USP28 abundance or activity suppresses growth and survival of 60
transformed lung cells. Furthermore, inhibition of USP28 via a small molecule 61
inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and 62
its inhibition cooperated with clinically established compounds used to target 63
EGFR
L858R
, BRAF
V600E
or PI3K
H1047R
driven tumor cells. Targeting USP28 protein 64
abundance already at an early stage via inhibition of its activity therefore is a feasible 65
strategy for the treatment of early stage lung tumours and the observed synergism 66
with current standard of care inhibitors holds the potential for improved targeting of 67
established tumors. 68
69
70
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Introduction 71
72
In the past decade, with the advent of targeted therapy, great advancements towards 73
the treatment of progressed Non-Small Cell Lung Cancer (NSCLC) in distinct patient 74
cohorts were achieved(1), while patients with early disease do not benefit from these 75
new treatments(2, 3). For this cohort, the curative treatment, still today, is the 76
surgical resection of a lung lobe. This is a severe procedure, inflicting major damage, 77
requires an extended recovery time and can result in therapy induced mortality(4, 5). 78
Furthermore, therapy failure in late stage tumours by establishment of treatment 79
escape mechanisms is a common observation in NSCLC, significantly affecting 80
patient survival(6, 7). Overall, survival rates have only marginally improved and most 81
patients still succumb to the disease(1). 82
83
Therefore, targeting of common essential pathways and exploiting tumour intrinsic 84
vulnerabilities holds the potential to not only improve current treatment for late stage, 85
but also for early stage patients. One central cellular component tumour cells alter 86
during oncogenic transformation is the ubiquitin proteasome system (UPS)(8, 9). The 87
dysregulation of the UPS is a prerequisite for tumor cells to tolerate increased 88
proliferation, metabolic changes, immune evasion and proteostatic stress 89
management(10). All these processes are ‘hallmarks of cancer’ and therefore 90
significantly contribute to disease progression, therapy failure and shorted survival. 91
Therefore, cancer cells, when compared to non-transformed cells, are dependent on 92
the ubiquitin system(11, 12). As a consequence, tumour cells develop exploitable 93
dependencies towards the expression and abundance of discreet members of the 94
UPS
12
. 95
96
Despite the prominent involvement of the UPS in cancer, our understanding of how 97
tumour cells alter the UPS system very early in transformation is rather limited(12). 98
The identification of essential and druggable key-players within this class of enzymes 99
has the potential to hold novel therapeutic strategies. Deubiquitinating enzymes are 100
such a therapeutically promising class of enzymes, as individual members can be 101
targeted by small molecule inhibitors(13-15). 102
103
In this study, we report that the deubiquitylase USP28 presents a UPS enzyme, 104
which is commonly upregulated during early stages of oncogenic transformation in 105
lung cancer. Irrespective of oncogenic driver, tumour cells upregulate USP28, which 106
stabilizes proto-oncogenes, such as c-MYC, c-JUN or NOTCH. Tumour cells are 107
addicted to USP28 to allow oncogenic transformation and its inhibition via the small 108
molecule inhibitor AZ1(16) partially reverts the oncogenic transformation. Finally, 109
combining USP28 targeting with targeted therapy against commonly found 110
oncogenic drivers potentiates treatment responses, at least in cellulo, indicating that 111
the UPS system, exemplified by USP28, is a promising target structure for lung 112
cancer. 113
114
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115
Materials and Methods 116
117
Cell lines 118
119
Human basal bronchial epithelial BEAS-2B cells were originally transformed with 120
SV40-large-T-antigen (Reddel et al., 1988). The cell line BEAS-2B was a kind gift of 121
Prof. Marco A. Calzado Canales (Universidad de Córdoba, Hospital Reina Sofia, 122
Córdoba, Spain).
BEAS-2B Oncogenic cells were generated upon retroviral infection 123
of BEAS-2B DIF with the next plasmids: EGFR (addgene number: #11011), EGFR 124
L858R (addgene number: #11012), pBabe puro HA PIK3CA (addgene number: 125
#12522), pBabe puro HA PIK3CA H1047R (addgene number: #12524), pBabe puro 126
HA PIK3CA E545K (addgene number: #12525), pBabe puro HRAS G12D (HRAS 127
G12D was cloned into pBabe puro in our lab) and pBabe puro BRAF V600E 128
(addgene number: #15269). The plasmids EGFR and EGFR L858R were a gift from 129
Matthew Meyerson (Greulich H, Chen TH et al. 2005). pBabe puro HA PIK3CA 130
H1047R, HA PIK3CA E545K, HA PIK3CA were a gift from Jean Zhao (Zhao JJ, Liu 131
Z, Wang L et al. 2005). pBabe Puro BRAF V600E was a gift from William Hah 132
(Boehm et al Cell. 2007) For virus production HEK293-T cells were used. Cell lines 133
used in this publication are listed in the supplementary table called: Consumables 134
and resources. 135
136
Tissue culture reagents and drugs.
137
138
Cells were plated on Greiner dishes and incubated at 37 °C, 95 % relative humidity 139
and 5 % CO2 in a cell incubator for optimal growth conditions. DIF BEAS-2B, 140
oncogenic BEAS-2B and HEK-293T cells were cultured in DMEM (Gibco) 141
supplemented with 10% fetal bovine serum (FCS)/ 1% Pen-Strep. UD cells were 142
cultured in LHC-9 (Gibco) supplemented with 1% Pen/Strep. To cultivate UD BEAS-143
2B cells, the dishes were pre-coated with pre-coating solution composed by: 0.03% 144
Collagen (in 0.1 M acetic acid), 0.01% Fibronectin and 0.001% BSA. UD cells were 145
supplemented with 10% FCS to induce pre-oncogenic differentiation. Cells were 146
routinely tested for mycoplasma via PCR.The reagents and drugs were dissolved in 147
Dimethyl sulfoxide (DMSO). AZ1, Gefitinib, Buparlisib and Vemurafenib were 148
purchased from Selleckchem. Drugs and reagents are listed in the supplementary 149
table called: Consumables and resources. 150
151
AAV, Retrovirus and Lentivirus production and purification 152
153
Adeno-associated viruses (AAVs) were generated and packaged in HEK293
‐
T cells 154
seeded in 15
‐
cm cell culture dishes (60-70% confluence). Cells were transfected 155
with the plasmid of interest (10
μ
g), pHelper (15
μ
g) and pAAV
‐
DJ (10
μ
g) using PEI 156
in ratio 2:1 (70
μ
g). After 96 hours, AAV Virus isolation from cells was performed as 157
previously described (17). For Retrovirus production, HEK293 cells (70% 158
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(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
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confluence) were transfected with the babe plasmid of interest (15
μ
g), pUMVC (10 159
μ
g) and VSV-G (10
μ
g) using PEI (70
μ
g). After 96 H, the medium containing retrovirus 160
was filtered (0.45 µM) and stored at -80°C. For Lentivirus production, HEK293 cells 161
(70% confluence) were transfected with the plasmid of interest (15
μ
g), pPAX (10 162
μ
g) and pPMD2 (10
μ
g) using PEI (70
μ
g). After 96 H, the medium containing 163
lentivirus was filtered (0.45 µM) and stored at -80°C. 164
165
In vitro DNA transfection and infection 166
167
DNA transfection was performed exposing 60% confluence BEAS-2B cells plated in 168
a 6-well cell culture dish to a mix of 2.5
μ
g plasmid of interest, 200
μ
l DMEM free 169
serum and 5
μ
l PEI (1:2 ratio). Upon 6h incubation at 37°C, 5% CO2 and 95% 170
relative humidity, the medium was removed and substituted by DMEM (Gibco) 171
supplemented with 10% FCS/ 1% Pen-Strep. For viral infection, 10 MOI (multiplicity 172
of infection) of Retroviruses (LVs) were added to normal medium of the cells in the 173
presence of polybrene (5
μ
g/ml). Cells exposed to the viruses were incubated at 174
37°C, 5% CO2 and 95% relative humidity for 4 days. The infected cells were 175
identified and selected by exposure to 2.5
μ
g/ml Puromycin for 72h. 176
177
RT-PCR 178
179
RNA was isolated with Peq GOLD Trifast (Peqlab), as indicated in the 180
manufacturer’s instructions. RNA was reverse transcribed into cDNA using random 181
hexanucleotide primers and M-MLV enzyme (Promega). Quantitative RT-PCR was 182
performed with SYBR Green mix (ABgene) on the instrument ´´Step One Realtime 183
Cycler´´(ABgene) The RT-PCR program employed in this research is the following: 184
95°C for 15 min., 40x [95°C for 15 sec., 60°C for 20 sec. and 72°C for 15 sec.], 95°C 185
for 15 sec. and 60°C for 60 sec. Relative expression was generally calculated with 186
ΔΔ
Ct relative quantification method. Melt curve was performed for all primers. For 187
visualization purposes, Excel (Microsoft) and Affinity Designer were used as 188
bioinformatic tools. Primers used for this publication are listed in the supplementary 189
table called: Consumables and resources. 190
191
Plasmids, sgRNA and shRNA Design 192
193
sgRNAs were designed using the CRISPR online tool: https://zlab.bio/guide-design-
194
resources
). shRNAs were designed using SPLASH-algorithm: 195
http://splashrna.mskcc.org/
) or RNAi Consortium/Broad Institute: 196
www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library
. 197
Oligonucleotides used in this publication are listed in the supplementary table called: 198
Consumables and resources. 199
200
Operetta analysis, Immunofluorescence, cell viability, Bliss synergy and GI50 201
202
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