Tumor Biology and Immunology

HER2 Amplification in Tumors Activates PI3K/Akt Signaling Independent of HER3 Ana Ruiz-Saenz, Courtney Dreyer, Marcia R. Campbell, Veronica Steri, Nate Gulizia, and Mark M. Moasser

Abstract

Current evidence suggests that HER2-driven tumorigenesis requires HER3. This is likely due to the unique ability of HER3 to activate PI3K/Akt pathway signaling, which is not directly accessible to HER2. By genetic elimination of HER3 or shRNA knockdown of HER3 in HER2-amplified cancer cells, we find residual HER2-driven activation of PI3K/Akt pathway signaling that is driven by HER2 through direct and indirect mechanisms. Indirect mechanisms involved second messenger pathways, including Ras or Grb2. Direct binding of HER2 to PI3K occurred through p-Tyr1139, which has a weak affinity for PI3K but becomes significant at very high expression and phosphorylation. Mutation of Y1139 impaired the tumorigenic competency of HER2. Total elimination of HER3 expression in HCC1569 HER2-amplified cancer cells significantly impaired

tumorigenicity only transiently, overcome by subsequent increases in HER2 expression and phosphorylation with bind- ing and activation of PI3K. In contrast to activation of onco- genes by mutation, activation by overexpression was quantita- tive in nature: weak intrinsic activities were strengthened by overexpression, with additional gains observed through further increases in expression. Collectively, these data show that progressive functional gains by HER2 can increase its repertoire of activities such as the activation of PI3K and overcome its dependency on HER3.

Significance: The intrinsic ability of HER2 to activate PI3K correlates with increased HER2 expression and can supplant the dependency upon HER3 for growth in HER2-amplified cancers. Cancer Res; 78(13); 3645–58. !2018 AACR.

Introduction Amplification and overexpression of HER2 are seen in a subset

of cancers including more than 20% of breast cancers. A consid- erable body of experimental evidence strongly suggests that HER2 is the oncogenic driver of these cancers, important in the genesis and progression of these tumors (reviewed in refs. 1, 2). HER2 belongs to a homologous family of receptor tyrosine kinases (RTK) that include EGFR, HER3, and HER4 (reviewed in ref. 3). Cooperation manifest as heterodimerization and transphosphor- ylation is a key feature of HER family signaling. Although these receptors are highly homologous in their extracellular ligand- binding and intracellular kinase domains, they are divergent in their intracellular c-terminal signaling tails. This allows for sig- naling via diverse effector pathways depending on the precise repertoire of HER family members expressed in a certain cell type and phosphorylated through heterodimerization events initiated by specific ligands. In particular, HER3 is uniquely endowed with 6 consensus binding site phosphotyrosines for the recruitment of

the SH2 domain of the regulatory subunit of PI3K, and is a potent activator of PI3K when phosphorylated (4, 5). Although EGFR and HER4 can directly or indirectly bind and activate PI3K (6, 7), the transmission of the signaling down the PI3K/Akt pathway of HER family members is typically thought to be mediated through the transphosphorylation of HER3.

As the role of the PI3K/Akt pathway in regulating numerous cellular processes paramount for tumorigenic growth has become more and more recognized, so has the role of HER3 as an upstream driver of this pathway. Experimental studies of the past decade have identified HER3 as a critical partner for HER2 in the genesis and progression of HER2-amplified cancers. Several knockdown or gene deletion models show that HER2-driven tumors do not develop or grow in the absence of HER3 expression (8–10). Naturally, this requirement for HER3 has been attributed to its potent ability to activate PI3K pathway signaling, which is critical for tumorigenic growth, and which HER2 is thought unable to directly activate.

We have been interested in developing deeper insights into the role of HER3 in HER2-amplified cancers. In particular, we have been interested in understanding why HER3 is required, whether the requirement is for the activation of PI3K, and whether its requirement is absolute or perhaps conditional or transient. This question is of paramount importance, as much effort is currently being placed into the development of HER3 inhibitors to com- plement HER2 inhibitors, and it would be of much value to know whether this will constitute the endgame in the treatment of HER2-amplified cancers, or whether it will provide benefits that are incremental in nature. Toward this cause, we engineered the loss of HER3 expression in HER2-amplified cancer cells and studied the resulting phenotypic consequences. The imme- diate findings show the loss of tumorigenic growth, confirming

Department of Medicine, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Corresponding Authors: Mark M. Moasser, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, Box 1387, San Francisco, CA 94143-1387. Phone: 415-476-0158; Fax: 415-353-9592; E-mail: [email protected]; and Ana Ruiz-Saenz, Phone: 415-476-0685; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-0430

!2018 American Association for Cancer Research.

Cancer Research

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previous reports. But this appears to be a transient phenotype, and increased HER2 expression and autophosphorylation ultimately supplants the requirement for HER3 and restores tumorigenic growth in a HER3-independent manner.

Materials and Methods Cells and reagents

Cell lines (BT474, SkBr3, HCC1569, CHO-K1, and MCF10A) were obtained from the ATCC. Authentication was performed using DNA Diagnostics Center. Cells were cultured at 37!C in a 95% air/5% CO2 atmosphere. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM)/HamF12 media (BT474 and SkBr3), RPMI 1640 media (HCC1569) or F-12K (CHO-K1) supplemented with 10% fetal bovine serum, penicillin, strepto- mycin, and L-glutamine. MCF10A cells were grown in DMEM/ HamF12 media containing 5% horse serum, 10 mg/mL insulin, 0.5 mg/mL hydrocortisone, 20 ng/mL EGF, penicillin, streptomy- cin, and L-glutamine. NR6cells were grown in DMEM highglucose containing 10% fetal bovine serum, sodium pyruvate, nonessen- tial aminoacids, 10 mmol/L HEPES buffer, penicillin, streptomy- cin, and L-glutamine. Conditioned medium from cell lines was routinely checked for mycoplasma contamination by PCR using the primers: 50 – GGG AGC AAA CAG CAT TAG ATA CCC – 30 and 50 – TGC ACC ATC TGT CAC TCT GTT AAC CTC – 30. Cells were used after a low number of passages (2–4) after thawing. Lapa- tinib was purified from commercial Tykerb tablets by organic extraction as previously described (11). GDC-0941 and BEZ235 were purchased from Selleckchem. Methyl Cellulose was pur- chased from Sigma-Aldrich (#M0262-250G). Matrigel Basement Membrane Matrix was purchased from Corning (Phenol Red-Free #356237).

DNA construction The HER2 mutants were generated at Genewiz and all mutated

vectors were confirmed by sequencing the entire cDNA insert. The Tyr 1143 to Phe mutation in NeuT was introduced using the Stratagene site-directed mutagenesis kit and protocol with the following complementary primers; the sense primer was 50- CCCAGCCCGAGTTTGTGAACCAATC-30, while the anti-sense primer was 50-GATGGTTCACAAACTCGGGCTGGG-30. For expression in CHO-K1 cells, HER2 wt and mutant cDNAs were HA tagged at the C-terminus and subcloned into the same mammalian expression vector. For expression in the MCF-10A cells, both NeuT and NeuTY1143F mutants were flag tagged at the C-terminus and subcloned into the same retroviral vector. Plas- mids used are as follows. Gene expression in NR6 cells and MCF10A was done using the retroviral pMSCV PIG vector (gift of David Bartel, addgene #21654). Human p85 expression in CHO-K1 cells was done using pSV-human p85a (gift of Ronald Kahn, addgene #11499). HER2 mutant expression in CHO-K1 cells was done using the pDEST40 plasmid vector (Thermo Fisher Scientific).

Cell transfection, retrovirus production, and production of stable cell lines

siRNA and plasmid transfections were performed using Lipofectamine 2000 according to the manufacturer's instruc- tions (Invitrogen). Cells were processed 24 or 72 hours after plasmid or siRNA transfection, respectively. Control siRNAs and siRNAs targeting H-Ras, N-Ras, K-Ras, EGFR, and Grb2

were obtained from Dharmacon. Cells transfected with control siRNAs were used as controls in all experiments.

For inducible knockdowns of HER3, the oligonucleotides were synthesized and annealed and then ligated into the pSuperior vector. Standard protocols were used according to the manufac- turer's (OligoEngine) instructions. shRNA sequences used are described below:

50-GATCCCAAGAGGATGTCAACGGTTATTCAAGAGATAACC- GTTGACATCCTCTTTTTTTA-30

50-AGCTTAAAAAAAGAGGATGTCAACGGTTATCTCTTGAATA- ACCGTTGACATCCTCTTGG-30

The NR6 and MCF-10A cell lines stably expressing HER2, NeuT or NeuTY1143F proteins were produced by infection with respec- tive retroviruses in the presence of 1 mg/mL polybrene (Sigma- Aldrich). After incubation in growth medium for approximately 48 hours, cells were treated with 1 mg/mL puromycin, the selection antibiotic expressed by the viral vector, to remove nonexpressing cells. Puromycin-resistant cell populations were used for the various experiments described in the relevant sections.

For HCC1569 HER3KO cells generation, CRISPR/Cas9 KO plasmids of HER3 (h, sc-400146) were used to generate stable HER3 knockout HCC1569 cells according to the manufacturer's instructions. Either individual or pooled single-cell clones with efficient target gene KO (as indicated in Figures) were used for downstream functional experiment to minimize the clonal variation.

Antibodies and Western blotting Cell and tumor lysates were prepared using modified RIPA lysis

buffer (150 mmol/L NaCl, 0.1% SDS, 1% Nonidet P40, 1% sodium deoxycholateand10mmol/Lsodium phosphate, pH7.2) supplemented with protease and phosphatase inhibitors. Tumor lysates were briefly sonicated to break chromosomal DNA that could interfere with polyacrylamide gel electrophoresis separa- tion of proteins. After clearing by centrifugation at 12,000 rpm for 10 minutes, the lysates were analyzed as described in the respec- tive experiments. For electrophoresis gel separation of proteins, samples were denatured by boiling with Laemmli sample buffer and run on an 8% denaturing polyacrylamide gel. After transfer onto a nitrocellulose membrane and blocking with 3% bovine serum albumin, the membranes were stained with primary anti- bodies overnight at 4!C, washed 3 times with TBST (Tris-buffered saline containing 1% Triton-X-100), stained with secondary anti- bodies for 1 hour at room temperature and detected using the chemiluminescence method. Western blots were performed using antibodies purchased from Santa Cruz Biotechnology (HER3 (5A12) #81455, actin #1616, vimentin (V9) #6260), Cell Signaling Technology (EGFR #2232, p-HER2Y1248 #2247, p- HER2pY1221/Y1222 #2243, pAKT-Thr308 #4056, pAKT-Ser473 #4058, p-MAPK #9101, MAPK #9102, Ras #3965, Grb2 #3972), Abcam (p-HER2Y1139 #53290), Bethyl (p85b #A302-593A), and Millipore (p85a #05-212). Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (mouse and goat) and GE Healthcare (rabbit).

Phospho-RTK profiling was done with the RayBio C-Series Phospho-RTK Antibody Array according to the manufacturer's instructions. Briefly, the array membranes were blocked in block- ing buffer and incubated with diluted cell lysates overnight at 4C. After washing, the array membranes were incubated with the detection antibody, washed again, and developed with standard chemiluminescent reagents.

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qPCR Total cellular RNA was isolated using the Qiagen RNeasy kit

following the manufacturer's instructions. RT and PCR ampli- fication of EGFR was performed using primers and methods that have been described previously (11). Quantitative PCR assays were performed for EGFR and the housekeeping gene b2- microglobulin from cDNA prepared from different mouse cell lines and tissue. We used the DDCT methodology to obtain the ratios after normalizing to b2-microglobulin. The ratios were obtained against the average for the positive control (cerebel- lum tissue). The mean ratio was determined on the duplicate values obtained.

Anchorage-independent growth in soft agar Anchorage-independent growth in the soft agar was used as one

of the assays to determine state of cell transformation. First, 6-cm cell culture plates were overlaid with 0.7% agar in the growth medium. Next, 1 " 104 cells/well of control or overexpressing HER2 NR6 cells, 1 " 104 cells/well of HCC1569-HER3WT or HCC1569-HER3KO in complete medium, and 5 " 103 cells/well of NeuT or NeuTY1143F-overexpressing MCF10A cells in medi- um without EGF and insulin, were suspended in 1.5 mL of growth medium mixed with melted agar to a final concentration of 0.4% and immediately poured onto the agar overlay. After 5 minutes of incubation at room temperature for the agar to solidify, the cells were transferred to a 37!C and 5% CO2 incubator. Colonies were counted after 10 days (MCF10A) or 21 days (Nr6 and HCC1569 cells) and pictures were taken using Quantity One software from Bio-Rad.

Coimmunoprecipitation Cells serum starved overnight and treated with 1 mmol/L

lapatinib or DMSO for 2 hours were scraped from the plate using RIPA (HCC1569 and BT474) or NP-40 (SkBr3) lysis buffer, incubated on ice for 30 minutes and centrifuged in a Minifuge for 5 minutes at 4!C. The supernatant was used for the immu- noprecipitation assay and a fraction of it as input. Lysates pre- pared from these cells were subjected to immunoprecipitation with 10 mg of trastuzumab coupled to protein G sepharose beads overnight at 4!C, washed 3 times with lysis buffer, eluted by boiling for 5 minutes with Laemmli sample buffer, and analyzed by immunoblotting as described above.

Animal experiments All mice studies were reviewed and approved by the UCSF

Institutional Animal Care and Use Committee. A total of 2 " 106 HCC1569 cells (100 mL, 1:1 matrigel: serum-free RPMI) were implanted subcutaneously in female nude mice (Foxn1nu/ Foxn1nu) at 6 to 8 weeks of age. For lapatinib treatment, when tumors reached a volume of 200 mm3, HCC1569 HER3KO- bearing mice were randomly split into two groups. Mice were treated daily by oral gavage either with lapatinb (100 mg/kg/day) or vehicle MCT control (0.5% w/v Methyl Cellulose, 0.2% v/v Tween 80). A total of 2 " 106 MCF10A cells (50 mL, 1:1 matrigel: serum-free RPMI) were implanted into the fourth mammary fat pad of female NSG mice (NOD-scid IL2Rg-null) at 6 to 8 weeks of age. Age-matched mice were assigned randomly to experimental groups. Biological replicates for each group are indicated by "n" in the figure legends of Figs. 1 and 6. Tumor growth was assessed weekly by caliper measurement.

Ki-67 staining Freshly harvested tumor tissue was fixed in 10% neutral buff-

ered formalin phosphate for 24 hours at 4!C. Tumor tissue was paraffin-embedded and sectioned 4 mm thick. For Ki-67 staining, paraffin tissue slides were heated in 60!C oven for 30 minutes, then deparaffinized by immersing the slides in xylene and rehy- drating the tissue in 100% ethanol, 95% ethanol, 70% ethanol and deionized water washing steps (5 minutes each). Slides were subsequently immersed in 3% H2O2 (in PBS) for 10 minutes to quench endogenous peroxidases. Both enzymatic and heat induced-antigen retrieval were performed as described: slides were immersed in prewarmed 0.25% Trypsin (in PBS) for 15 minutes, washed in PBS for 5 minutes, then immersed in 10 mmol/L Citrate Buffer and heated in microwave oven (900 W) for 10 minutes. Slides were let cool down at room temperature for 30 minutes and blocked with 1.5% Normal Goat Serum (in PBS) for 30 minutes at room temperature. Slides were stained with monoclonal mouse a-human Ki-67 Antigen antibody (1:200 in PBS 0.05% Tween-20; clone MIB-1, #M7240, Dako) overnight at 4!C. Following primary antibody incubation, slides were extensively washed in PBS. As secondary antibody, horse a-mouse-biotinylated antibody 1:200 in PBS 0.05% Tween-20, #BA-2000, Vector Laboratories) was incubated for 30 minutes at room temperature followed by Vectastain Elite ABC HRP kit (#PK- 6100, Vector Laboratories). Slides were washed in PBS and 3,30- diaminobenzidine tetrahydrochloride (DAB) substrate (#D5905, Sigma-Aldrich) was added according to the manufacturer's instructions. Nuclear hematoxylin counterstain was applied, followed by dehydration through 70% ethanol, 95% ethanol, 100% ethanol, and xylene (3 minutes each). Slides were mounted with Permount Mounting Medium (Thermo Fisher Scientific) and analyzed on a bright-field microscope.

Fluorescent in situ hybridization for HER2 Fluorescent in situ hybridization (FISH) was performed using

the PathVysion (Vysis Inc.) probe kit. In brief, tumor tissue was paraffin-embedded and sectioned 4 mm thick. The section was air dried, pretreated, and digested with proteinase K before being hybridized with fluorescent-labeled probes for the HER2 gene, and a-satellite DNA for chromosome 17. Nuclei were routinely counter- stained with an intercalating fluorescent counterstain, DAPI. Tumors were imaged using a Spinning disk confocal Nikon TI inverted microscope ("60 objective, lasers 640, 488, and 561 nm). For each tumor, at least 43 tumor cell nuclei were counted and scored for both HER2 and chromosome 17 centromere numbers.

Quantification and statistical analysis For all analyses, significance was determined at P < 0.05. All

analyses, unless specified elsewhere, were performed in GraphPad Prism (GraphPad Software, Inc.). For analyses limited to two groups, Student t test was utilized. Data are expressed as mean # 1 SEM, unless otherwise specified. For each experiment, unless otherwise noted, n represents the number of individual biological replicates.

Results HER2-amplified cancer cells regrow following HER3 knockout

We have been interested in the role of HER3 in mediating HER2-driven tumorigenesis. Using CRISPR-Cas9 technology, we engineered the elimination of HER3 expression in HER2-

HER2-Amplified Tumors Overcome the Requirement for HER3

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amplified HCC1569 breast cancer cells. Three independent HER3 knockout (HCC1569-HER3KO) clones, confirmed to lack HER3 expression, were selected for further analysis (Fig. 1A). Three clones that failed to undergo the gene targeting event and main- tained HER3 expression were also selected as controls (HCC1569- HER3WT). HCC1569-HER3KO cells maintain their proliferative growth and can be maintained in continuous monolayer cell culture. In three-dimensional culture conditions, both HCC1569- HER3WT and HCC1569-HER3KO formed solid acini although HER3 depletion significantly decreased the size and number of HCC1569-HER3KO acini, in agreement with previous studies (Supplementary Fig. S1A and S1B; ref. 9). To determine the effect of HER3 function on tumorigenic growth, the three HCC1569- HER3WT and three HCC1569-HER3KO cells were implanted in nude mice. HCC1569-HER3WT cells generated tumors within 2 weeks and with a high take rate, whereas HCC1569-HER3KO cells failed to generate any tumors despite 14 weeks of follow-up (Fig. 1B). However, with continuous follow-up of mice for durations approaching 1 year, tumors did eventually develop in about two- thirds of the HCC1569-HER3KO inoculated mice (Fig. 1B). Tumors from these mice were surgically excised at necropsy for further analysis to gain insights into the mechanisms underlying the restoration of their tumorigenic properties. Tumor regrowth was not due to restoration of HER3 expression, as confirmed by Western blot analysis of tumor lysates. Interestingly, we found that HER2 expression and phosphorylation were increased in HCC1569-HER3KO tumors (Fig. 1C and D). This was not due to an increase in HER2 gene copy numbers as determined by FISH analysis of tumor blocks (Supplementary Fig. S1C). Receptor tyrosine kinase (RTK) profiling blots were conducted on selected tumor lysates to determine whether phosphorylation of other RTKs has been induced in compensation for the loss of HER3 phosphorylation, but no such compensatory RTKs were evident besides HER2 phosphorylation (Fig. 1E; Supplementary Fig. S2A and S2B). To determine whether other oncogenic pathways may have supplanted the oncogene HER2, these tumors were re- implanted in mice and treated with the HER2 inhibitor lapatinib. Lapatinib inhibits the growth of these HER3-null tumors, con- firming that they remain driven by HER2 (Fig. 1F; Supplementary Fig. S3). These HCC1569-HER3KO tumors display a lapatinib sensitivity that is nearly identical to the sensitivity of the parental HCC1569 tumors, which we have previously published (11).

HER3-null HER2-amplified cancer cells can have active Akt signaling

In the analysis of HER2–HER3 and downstream signaling in these tumors, it was apparent that the HCC1569-HER3KO tumors

have a further increase in expression of already overexpressed HER2, accompanied by further increase in autophosphorylation of HER2, in comparison with HCC1569-HER3WT controls (Fig. 1C and D). Differences in tumor cellularity were accounted for by a vimentin blot that is specific for human vimentin and noncross reactive with mouse vimentin (confirmed in Supplementary Fig. S4), making it easier to interpret the data from the low cellularity T1 tumor. With this in mind, it is evident that MAPK signaling is increased in most tumors above control cells. This could be a consequence of the observed increase in HER2 autophosphoryla- tion. Alternatively observed increase in HER2 phosphorylation may be a consequence of a primary increase in MAPK activity, with consequential effects on HER2 trafficking or membrane stability or phosphorylation half-life. The observations with Akt signaling were more unexpected and reflects diverse mechanisms used for bypassing the requirement for HER3. Some HCC1569-HER3KO tumors show a reduction or loss of Akt signaling, expected from their lack of HER3 function, which is recognized as the predom- inant upstream effector of Akt signaling in HER2-amplified can- cers. However, other tumors have restoration of Akt signaling despite the complete absence of HER3 expression. This suggests that HER2, when overexpressed, may be competent at activating Akt in a HER3-independent manner. Akt activation in HCC1569- HER3KO cells is driven by HER2, because it is lost with lapatinib treatment (Fig. 1G).

HER2–Akt signaling is preserved in HER3 knockdown cells Although the evidence from the recurrent HCC1569-HER3KO

tumors shows that the requirement for HER3 can be supplanted through diverse mechanisms, the one possibility that HER2 could activate PI3K directly was intriguing and surprising, and thus we pursued a series of experiments to more directly explore this potential activity in HER2.

For this, we used NR6 cells, which lack the expression of the entire HER family of RTKs (confirmed in Supplementary Fig. S5; ref. 12). Stable overexpression of HER2 in NR6 cells leads to cell transformation despite the lack of HER3 or any other HER family partners in these cells (Fig. 2A). These HER2-transformed cells show increased MAPK and Akt signaling compared with controls (Fig. 2B and C). The increased MAPK and Akt signaling is attrib- utable to upstream HER2 because it is suppressed by lapatinib. The increased Akt signaling is mediated through PI3K, because it is suppressed by PI3K inhibitors (Fig. 2D). The activation of Akt in these cells is not due to a secondary induction of HER3 that is a consequence of HER2 transformation, as HER3 remains silent in these cells following HER2 transformation (Supplementary Fig. S5). This evidence supports the hypothesis that HER2, when

Figure 1. HER3 knockout in HER2-amplified cancer cells transiently suppresses tumor growth. HCC1569 cells underwent Crispr/Cas9 targeting of the ERBB3 gene. Three HER3-null clones were selected for further experiments (HER3KO), and three clones that failed to undergo ERBB3 gene targeting were selected for use as controls (HER3WT). A, Cell lysates were assayed by Western blotting as indicated, showing the loss of HER3 protein expression in the three HER3KO selected clones. B, Three clones from HCC1569-HER3WT and three clones from HCC1569-HER3KO cells were implanted subcutaneously into female nude mice (5 nude mice/clone; 2 " 106 cell/implant) and tumor volume was measured weekly. The different clones of HCC1569-HER3KO cells are shown in three different colors. C, Lysates from resected tumors were immunoblotted using antibodies against the indicated proteins. The species specificity of the anti-human vimentin was confirmed in Supplementary Fig. S4. D, The immunoblot signals from C were quantified and the relative levels of pHER2, pAkt, and pMAPK are shown here as indicated. Levels were normalized to actin. E, Lysates from a HCC1569-HER3WT tumor and three HCC1569-HER3KO tumors were assayed using a phospho-RTK profiling antibody array. The array legend is provided in Supplementary Fig. S2A. The rectangles indicate the duplicate dots corresponding to p-HER2. F, An HCC1569-HER3KO tumor was resected and re-implanted in 10 nude mice. When tumors reached a volume of 200 mm3, HCC1569-HER3KO-bearing mice were treated daily by oral gavage either with lapatinib at 100 mg/kg/day (n ¼ 5 mice) or vehicle control (n ¼ 5 mice) and tumor volumes were measured weekly. Mean tumor volumes are plotted with error bars showing SEM. The growth inhibition by lapatinib is significant at P < 0.01. G, HCC1569-HER3KO cells growing in culture were treated with 1 mmol/L lapatinib or DMSO for 2 hours and cell lysates studied by Western blotting as indicated.

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overexpressed, can activate Akt and induce cell transformation without a requirement for its heterodimeric partners including HER3.

Because HER2 appears capable of activating PI3K without its HER family partners, we next asked whether some of the PI3K/Akt signaling in HER2-amplified cancer cells may be directly mediated by HER2 rather than through HER3. This was done by engineering

HER2-amplified cancer cell lines with doxycycline-inducible shRNA knockdown of HER3 expression. In three different exam- ples of HER2-amplified cancer cells, it is evident that substantial Akt signaling persists despite near complete suppression of HER3 expression (Fig. 2E). The observed Akt signaling in the absence of HER3 is driven by HER2, not other receptor families, as it is lost following lapatinib treatment (Fig. 2E).

Figure 2. HER2 overexpression activates Akt without a requirement for HER3. A, NR6 cells stably transfected with HER2 or vector control were seeded in soft agar and colonies imaged and counted at 21 days. n ¼ 3; %, P < 0.03. B, Control and HER2-transfected NR6 cells were treated with 1 mmol/L lapatinib for 2 hours and cell lysates were assayed by Western blotting as indicated. C, Quantitation of pAkt signaling from the Western blots of B. Data were pooled from three (pAktT308) and five (pAktS473) independent experiments and are presented as the mean þ SEM. %, P < 0.03; %%, P < 0.01. D, Control and HER2-transfected NR6 cells were treated with 0.5 mmol/L GDC-0941 and 0.25 mmol/L BEZ235 for 1 hour and cell lysates were assayed by Western blotting as indicated. E, HER2-overexpressing HCC1569, BT474, and SkBr3 cells engineered to induce expression of a HER3 shRNA in a doxycycline-inducible manner were treated with 100 ng/mL doxycycline for 72 hours (HCC1569 and BT474) or 7 days (SkBr3) and also with 1 mmol/L lapatinib for 2 hours before cell harvest. Cell lysates were assayed by Western blotting as indicated.

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HER3-independent Akt activation does not require Ras in HER2-amplified cancer cells.

To explore the mechanism by which HER2 mediates Akt activation in these tumor cells, we studied the contribution of the small GTPase Ras. Ras has been shown to directly interact with and activate class I PI3Ks through a Ras-binding domain (RBD) that is present in the p110 catalytic subunit of PI3K (13–15). This may provide an alternative and redundant pathway linking over- expressed HER2 with PI3K, which can account for the observed lapatinib-sensitive HER2–Akt signaling in HER2-amplified cancer cells. We subjected HCC1569TR-shHER3, BT474TR-shHER3, and SkBr3TR-shHER3 cells to the dox-induced depletion of HER3 and also the depletion of Ras using pooled siRNAs targeting H-Ras, N- Ras, and K-Ras. The depletion of HER3 and Ras proteins has a small effect on Akt signaling in two of these cell types, but substantial Akt signaling remains and is lapatinib suppressible, confirming that it is driven by HER2 in these cells (Fig. 3).

HER2 interaction with PI3K does not require partners or known adapters

To determine whether HER2 can itself recruit and activate PI3K, we looked for the p85 regulatory subunit of PI3K in HER2 immune complexes. This interaction was studied initially in the

simpler NR6 experimental cell system and subsequently in the more physiologic cancer cell context. In HER2-transformed NR6 cells, p85 is readily detected in HER2 immune complexes, and this interaction is lost when HER2 is dephosphorylated by lapatinib treatment (Fig. 4A). This interaction cannot be mediated through the HER family heterodimeric partners of HER2 as NR6 cells lack expression of the HER family proteins (Supplementary Fig. S5).

The interaction was then studied in HER2-amplified cancer cells. HCC1569TR-shHER3, BT474TR-shHER3, and SkBr3TR- shHER3 cells were depleted of HER3 expression by doxycycline treatment and the presence of p85 was clearly detected in HER2 immune complexes in these cancer cells (Fig. 4B). HER2–p85 interaction was also detected in HCC1569-HER3KO cells (Fig. 4C). The interaction requires HER2 phosphorylation and is lost following lapatinib treatment (Fig. 4A–C). The specific p85 isoform that is expressed and that is observed in HER2 immune complexes varies depending on the cell context and this was determined in an initial set of experiments (Supple- mentary Fig. S6A).

The HER2–PI3K interaction, in these cancer cells, may in theory be indirectly mediated through the other heterodimeric partners of HER2, either EGFR or HER4. The three cancer cell lines studied above lack expression of HER4, eliminating this as an

Figure 3. HER2 activation of Akt does not require Ras.A, Theindicated three engineered HER2-amplified cancer cell lineswere treated with 100 ng/mL doxycycline for 72 hours (HCC1569 and BT474) or 7 days (SkBr3) to knockdown HER3 expression, and transfected with a pool of siRNA Ctrl or a pool of siRNA against H-Ras, K-Ras, and N-Ras for 72 hours. Additional arms were also treated with 1 mmol/L lapatinib for 2 hours before cell harvest. Cell lysates were assayed by Western blotting as indicated. Values represent the intensities of pAkt bands relative to that of the corresponding total Akt band. The blots are representative of multiple independent experiments.

HER2-Amplified Tumors Overcome the Requirement for HER3

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intermediate, but they do express EGFR. EGFR lacks consensus phosphotyrosine-binding sites for p85, but can nevertheless bind and activate PI3K indirectly through Gab1 (6, 16). To address this, we subjected HCC1569TR-shHER3, BT474TR-shHER3, and SkBr3TR-shHER3 cells to the dox-induced depletion of HER3 and also the siRNA-induced depletion of EGFR. The interaction of p85 with HER2 was retained in these HER3-and EGFR-depleted cells, again suggesting a direct interaction (Fig. 5A). This interac- tion requires HER2 phosphorylation as it is lost upon lapatinib treatment (Fig. 5A).

HER2 activates the Ras/MAPK pathway via the recruitment of the adaptor proteins Grb2 and Shc to different tyrosines located in its C-terminal tail (17, 18). These adaptor proteins could poten- tially also be mediating the interaction with PI3K as has been shown for other tyrosine receptors kinases (19–21). To explore this possibility, we looked for those adaptor proteins in coimmu- noprecipitation assays. We detected Grb2, but not Shc, in HER2 immune complexes in the three HER2-amplified cancer cell lines (Fig. 5B). This interaction was also lapatinib sensitive. To deter- mine whether the HER2–p85 interaction was mediated through Grb2, we subjected HCC1569TR-shHER3, BT474TR-shHER3, and SkBr3TR-shHER3 cells to the dox-induced depletion of HER3 and

also the siRNA-induced depletion of Grb2. The depletion of HER3 and Grb2 proteins partially reduces the observed p85 protein in HER2 immune complexes in one of the three cell types (Fig. 5C). But residual HER2–p85 interaction is evident nevertheless. The interaction requires phosphorylation of HER2 as it is lost follow- ing lapatinib treatment. To validate that the interaction between HER2 and PI3K occurs in a Ras-independent manner, we per- formed coimmunoprecipitation assays in control and Ras-deplet- ed cells. HER2–PI3K complexes are preserved in tumor cells depleted of both HER3 and Ras proteins and these complexes remain lapatinib sensitive (Supplementary Fig. S6B).

HER2–Akt signaling is a product of massive HER2 expression The interaction of HER2 with PI3K is only seen in the con-

text of constitutive phosphorylation induced by massive HER2 expression such as when experimentally overexpressed in NR6 cells, or when overexpressed due to gene amplification in cancer cells. It is important to note that this interaction is not detected in the normal physiologic setting of signaling, such as in cells with normal levels of HER2 expression that are stimulated to induce HER2–HER3 phosphorylation and signaling by physiologic ligands (Fig. 5D).

Figure 4. Overexpressed HER2 can bind PI3K. A, Control and HER2-overexpressing NR6 cells were treated with 1 mmol/L lapatinib for 2 hours. Cell extracts were immunoprecipitated with anti-HER2 antibodies or control IgG. The immune complexes were analyzed by immunoblotting as indicated. B, The indicated HER2- amplified cancer cell lines were treated with 100 ng/mL doxycycline for 72 hours (HCC1569 and BT474) or 7 days (SkBr3) to knockdown HER3 expression. Additional arms were also treated with 1 mmol/L lapatinib for 2 hours before harvest. Anti-HER2 immunoprecipitates were immunoblotted as indicated. The p85 immunoblots are specifically p85a for HCC1569 and BT474 and p85b for SkBr3. C, HCC1569-HER3WT and HCC1569-HER3KO cells were treated with 1 mmol/L lapatinib for 2 hours and anti-HER2 immunoprecipitates were immunoblotted as indicated.

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HER2–PI3K interaction requires HER2–Y1139 phosphorylation

Because the intermediaries such as Ras or Grb2 could not entirely account for the HER2–PI3K interaction, we investi- gated the possibility that HER2 may engage PI3K directly. The

HER2 c-terminal tail has numerous tyrosines that are phosphor- ylated during physiologic ligand-driven receptor signaling. Some of these are also constitutively phosphorylated when HER2 is massively overexpressed such as in HER2-amplified cancer cells. In fact, the constitutive autophosphorylation of HER2 c-terminal

Figure 5. HER2 interaction with PI3K is not dependent on indirect binding partners. A, The indicated three engineered HER2-amplified cancer cell lines were treated with 100 ng/mL doxycycline for 72 hours (HCC1569 and BT474) or 7 days (SkBr3) to knockdown HER3 expression, and transfected with a pool of siRNA Ctrl or a pool of siRNA against EGFR for 72 hours. Additional arms were also treated with 1 mmol/L lapatinib for 2 hours before cell harvest. Anti-HER2 immunoprecipitates were assayed by Western blotting as indicated. The p85 immunoblots are specifically p85a for HCC1569 and BT474 and p85b for SkBr3. B, The same cell lines were depleted of HER3 expression by doxycyline treatment and anti-HER2 immunoprecipitates were assayed by Western blotting for the presence of Grb2. Additional arms were also treated with 1 mmol/L lapatinib for 2 hours before cell harvest. C, The same cell lines were depleted of HER3 expression by doxycyline treatment and transfected with a pool of siRNA Ctrl or a pool of siRNA against Grb2 for 72 hours. Additional arms were also treated with 1 mmol/L lapatinib for 2 hours before cell harvest. Anti-HER2 immunoprecipitates were assayed by Western blotting as indicated. The p85 immunoblots are specifically p85a for HCC1569 and BT474 and p85b for SkBr3. D, T47D breast cancer cells were treated with 1 mmol/L lapatinib for 2 hours and stimulated with 10 ng/mL heregulin for 15 minutes. Cell lysates were analyzed by immunoblotting as indicated (top) or immunoprecipitated with control IgG or anti-HER2 antibodies (bottom). The immunoprecipitates were analyzed by immunoblotting with anti-HER2 and anti-p85 antibodies as indicated.

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tyrosines is increased in HER3 knockout HCC1569 cancer cells, suggesting that they may play a role in compensation for the loss of HER3 (Fig. 1C; Supplementary Fig. S1A). A similar upregula- tion of HER2 phosphorylation becomes evident in HER3 shRNA knockdown cells if the knockdown is maintained for several weeks (Supplementary Fig. S7). A substantial conceptual barrier to the hypothesis that HER2 phosphotyrosines may directly engage PI3K is prior biochemical studies that have failed to identify such an interaction, and proteomic studies that have described consensus binding sites for the p85 subunit of PI3K on

the c-terminal tails of HER3 and HER4, but not on HER2 (22–24). However, these determinations have been made in the context of physiologic signaling and in cells with normal levels of expression of these proteins, in attempts to understand the basic mechanisms underlying cell signaling. The massive overexpression of HER2 that occurs in cancer cells may render significance to low affinity interactions that may be insignificant at normal expression levels and may have been dismissed in prior studies. In biochemical studies of peptides derived from the c-terminal tails of the HER family RTKs, the HER2 c-terminal tail indeed lacks high-affinity-

Figure 6. HER2 interaction with PI3K is mediated through tyr1139. A, HCC1569TR-shHER3 cells were treated with 100 ng/mL doxycycline for 72 hours to knockdown HER3 expression and the phosphorylation of HER2 c-terminal tail tyrosine residues was assayed by Western blotting using the indicated antibodies. An additional arm was treated with 1 mmol/L lapatinib for 2 hours prior to harvest. The p-HER2 bands were quantified by densitometry and graphically depicted (n ¼ 4). %, P < 0.03; %%, P < 0.01. B, CHO-K1 cells cotransfected withwild-type HER2 or a 4YF mutant of HER2 or vector control, aswell as human p85a and treated with 5 mmol/L lapatinib for 1.5 hours, and cell lysates analyzed by Western blotting as indicated. C, CHO-K1 cells were cotransfected with wild-type HER2 or a 4YF mutant of HER2 or vector control, as well as human p85a and anti-HER2 immunoprecipitates analyzed by anti-p85a immunoblotting. D, CHO-K1 cells were cotransfected with HER2 or each of 7 different mutant-HER2 constructs as well as human p85a. Anti-HER2 immunoprecipitates were analyzed by immunoblotting as indicated.

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binding sites for p85. However, several peptides from sites of tyrosine phosphorylation of HER2 were found to have low affinity and Y1139 had an intermediate affinity for the SH2 domain of p85 (25). Therefore, we interrogated whether these tyrosines on HER2 may be mediating the observed PI3K binding and signaling observed with overexpression of HER2. These tyrosines are indeed phosphorylated in HCC1569 cells and their phosphorylation increases over time when HER3 expression is knocked down (Figs. 1C and 6A; Supplementary Fig. S7). Initially, a quadruple YF mutant of HER2 was generated (HER2 4YF), mutated at Y1023, Y1139, Y1221, and Y1222, and was overexpressed in CHO-K1 cells, which have very low expression of HER2 and lack expression of the other members of the HER family (26, 27). While wild-type HER2 is observed to interact with p85 and induce the stimulation of Akt signaling above endogenous levels, this is substantially reduced in the HER2 4YF mutant (Fig. 6B and C). The p85 binding and Akt stimulation are linked with the autophosphorylation of

HER2 as they are eliminated following lapatinib treatment. To more specifically interrogate the role of the 4 individual tyrosines, we generated single and various combinations of double YF mutants and expressed them in CHO-K1 cells. The stability and expression of all these mutants were confirmed (Supplementary Fig. S8). The results from coimmunoprecipitations assays using these single- and double-mutant constructs identify that Y1139 is the critical tyrosine for p85 binding (Fig. 6D).

NeuT-Y1143F mutation reduces transformation and tumor growth

While the data above strongly suggest the direct activation of PI3K by HER2, they do not address its functional significance. To address this, we sought to determine whether this activity of HER2 is relevant to transformation or tumorigenesis. Because mutating all the copies of the HER2 amplicon in the existing HER2-amplified cancer cell lines is not technically feasible, we

Figure 7. The direct activation of PI3K by HER2/Neu contributes to tumorigenic activity. A, Human and mouse sequences showing the homology between human Y1139 and mouse Y1143 in the HER2 c-terminal tail. B, MCF10A cells stably expressing flag- tagged NeuT or NeuTY1143F were generated and immunoblotted as indicated. C and D, MCF10A-Ctrl, MCF10A-NeuT, and MCF10A- NeuTY1143F cells were seeded in soft agar and colonies imaged, counted, and colony diameters measured after 10 days. The differences in number of colonies (%, P < 0.01) and colony diameters (%, P < 0.01) are significant. Bars, 100 mm. E, NSG mice were injected with MCF10A cells stably expressing NeuT-Flag (n ¼ 11) or NeuT-Y1143F-Flag (n ¼ 10) in the mammary fat pad and tumor growth was measured weekly. Mean tumor volumes are shown with standard error of the mean (%, P < 0.016). Representative macroscopic images of isolated tumors showing the difference in size. Bars, 1 cm.

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tested the functional role of this tyrosine in experimental models of HER2/Neu-induced transformation and tumorigenesis. MCF10A-immortalized breast epithelial cells were transformed by NeuT, the rodent oncogenic homologue of HER2. The choice of NeuT over HER2 was due to its strongly transforming and tumorigenic properties in this experimental model, because human HER2 is only minimally transforming and nontumori- genic in these epithelial cells. As such MCF10A-NeuT cells are widely used as a surrogate for the study of HER2-driven transfor- mation (28–33). MCF10A cells were transfected with vector control, with NeuT, or with NeuT-Y1143F, the tyrosine homol- ogous to Y1139 in human HER2 (Fig. 7A) and similar expression levels of the two constructs confirmed (Fig. 7B). As expected, overexpression of NeuT induces a hyperactivation of Akt; how- ever, this activity is substantially reduced in the NeuT Y1143F

mutant (Fig. 7B). The role of downstream signaling pathways, other than PI3K, in mediating p-Y1143 signaling is difficult to know. In soft agar colony formation assays, NeuT is highly trans- forming, whereas the NeuT Y1143F mutant has 40% impairment in transformation efficiency and significantly decreased acini diameter (Fig. 7C and D). The tumorigenic activity of these NeuT constructs was compared by inoculating NSG mice with these MCF10A-transformed cells. Comparison of these experimental arms shows a clear impairment in the tumorigenic activity of NeuT imparted by a Y1143F mutation (Fig. 7E).

Discussion Understanding how HER2 promotes tumorigenic growth is a

subject of great interest that continues to evolve. The current state of our understanding suggests that HER3 is essential for HER2- driven tumorigenesis. This is based on HER3 knockdown studies in human cancer cell lines, and mammary ERBB3 gene ablation in a mouse genetic model of Neu-induced mammary tumorigenesis (8–10). Our findings do not refute these reports, but rather expand upon them. We confirm the suppression of tumor growth following HER3 knockout in HCC1569 cells, consistent with knockdowns reported in two other cell lines (8, 9). However, we report that this requirement can ultimately be overcome. The mouse mammary ERBB3 gene ablation is not directly comparable with HER3 knockout in a HER2-amplified cancer cell line because the former is a test of tumor genesis while the latter is a test of established tumors. HER3 or ERBB3 may yet have a requirement for the genesis of HER2 or Neu driven tumors that cannot be supplanted. However, the requirement in the case of established tumors is clearly supplantable as we have shown.

This work highlights important features of the oncogene HER2 that sets it apart from many other kinase oncogenes. The mode by which the HER2 proto-oncogene is converted into an oncogene is through gene amplification and protein overexpression. A gain- of-function incurred by amplification and overexpression, unlike gain-of-function incurred by point mutation or fusion events, is a progressive and quantitative mode of enhancing function. Certain functions may be gained at lower thresholds of overexpression, while additional functions may be gained at higher thresholds of overexpression. It appears that the HER2 oncoprotein, at over- expression and phosphorylation levels sufficient for cellular transformation, is unable to drive tumorigenesis by itself and requires a partner in HER3. However, at higher levels of HER2 phosphorylation, the requirement for HER3 can be overcome. An increase in HER2 phosphorylation can occur through further

increases in amplification levels, increases in HER2 transcription or protein half-life, increases in homodimerization events, or perhaps decreases in dephosphorylation activity. Such a volume knob attribute is not readily evident in kinase oncogenes activated through mutational events.

Quantitative functions gained through progressive increases in expression are likely to be related to increased interaction partners, conferring a degree of promiscuity to an otherwise highly specific and focused physiologic signaling network. Here, we show that a weak affinity interaction between phos- phorylated HER2 and PI3K becomes significant at very high expression thresholds. It is also possible that there are HER2 interactions with additional proteins that gain significance at very high expression levels, and these may also contribute to the tumorigenic properties of HER2 overexpression, including those that are redundant or nonredundant with HER3, and which may contribute to the initial HER2-induced tumorigen- esis or contribute to its escape from HER3 dependency. A protein microarray study on the HER signaling network and its interacting partners performed by Jonas and colleagues, 2006 revealed that HER3-interacting network changes very little as the affinity threshold of the interactions is lowered, whereas EGFR and HER2 become much more promiscuous (25). These data suggest that elevated levels of HER3 should primarily induce stronger signaling through pathways that are normally activated by low levels of HER3. In contrast, elevated levels of EGFR or HER2 should induce signaling through alternative pathways that are not activated at lower levels. The biological implication of this finding in the context of HER2 overexpres- sion is profound because a large subset of breast, esophageal, and gastric cancers show HER2 amplification or overexpression (34–39). It is possible that the high oncogenic potential of HER2 arises, at least in part, from its ability to turn on different pathways when overexpressed, rather than simply from stron- ger signaling through its primary pathways.

The repertoire of effector mechanisms engaged by HER2 may also be cell context dependent and we see evidence of this here as well. For example, the knockdown assays performed using siRNAs against Ras, EGFR, and Grb2 revealed that EGFR may contribute more significantly to PI3K/Akt activation in HCC1569 cells but not in BT474 and SkBr3 cells, Ras may contribute more to PI3K/Akt activation in HCC1569 and SkBr3 and less in BT474 cells, and Grb2 may partially mediate PI3K/ Akt signaling in BT474 cells but not in HCC1569 or SkBr3 cells. As such, it appears that HER2 can activate PI3K/Akt through a variety of direct and indirect effector mechanisms that may be available to it. Some tumors in our initial series developed HER3 independency without an obvious restoration of Akt signaling (Fig. 1C). Even these tumors remain driven by HER2 as evident by lapatinib treatment of one such tumor (Fig. 1F; Supplementary Fig. S3). The heterogeneity in effector signaling mechanisms is merely a consequence of the promiscuity that comes with massive overexpression. While such heterogeneity presents a daunting case for the selection of targeted therapies to be used in combination with HER2 inhibitors, it does portray a more singular role for HER2 as the oncogenic driver of this cancer subtype, and more promising expectations for future agents that can much more potently inactivate HER2.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

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Authors' Contributions Conception and design: A. Ruiz-Saenz, M.M. Moasser Development of methodology: A. Ruiz-Saenz, V. Steri, M.M. Moasser Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Ruiz-Saenz, C. Dreyer, M.R. Campbell, V. Steri, N.P. Gulizia, M.M. Moasser Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Ruiz-Saenz, N.P. Gulizia, M.M. Moasser Writing, review, and/or revision of the manuscript: A. Ruiz-Saenz, M.M. Moasser Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Ruiz-Saenz, M.R. Campbell, V. Steri Study supervision: A. Ruiz-Saenz, M.M. Moasser

Acknowledgments This work was supported in part by grants R01CA122216 and

U54CA112970 from the NIH. A. Ruiz-Saenz was supported in part by a

Ramon Areces Foundation postdoctoral fellowship grant. pMSCV NeuT was kindly provided by Dr. A Goga. We thank the members of the Preclinical Therapeutics Core at UCSF Helen Diller Family Comprehensive Cancer Center, in particular Julia Malato, for instruction and support for in vivo experiments; members of the Parnassus Flow Cytometry Core for cell sorting assistance (DRC Center Grant NIH P30 DK063720), Dr. Jean-Philippe Coppe for helpful discussions, and Dr. Elena Minones-Moyano for provid- ing mouse cerebellum tissue.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 7, 2018; revised April 10, 2018; accepted May 4, 2018; published first May 14, 2018.

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2. Abstract of proposed research project (not exceeding 750 characters)*

High-risk Human Papillomaviruses (HR HPVs) such as HPV16 infect epithelial cells including those of the genital tract and cause many cancers such as cervical cancer; due to the expression of E6 and E7 oncoproteins that attenuate tumour suppressor proteins p53 and pRb respectively. Cervical cancer affects millions of women and current treatment and diagnosis is limited. RNA aptamers are small, non-coding RNAs that bind proteins with high affinity and specificity. This research aims to screen RNA aptamers specific to E6 to determine if expression allows cervical cancer cells to undergo apoptosis. Aptamer sequences will be cloned and transfected into cells prior to analysis of apoptosis using propidium iodide and annexin V staining. This work may indicate the usefulness of RNA aptamers in the study of diseases and HR HPVs and improve diagnosis and treatment of HPV16 cancers.

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3. Description of research project (in no more than 6 sides of A4 describe the proposed research project providing a case for support. Font size Ariel 11pt or higher, 2 cm margins)

The case for support must describe:

a) A summary of the aims and objectives of the project and the importance of the research being proposed (UP TO 1 A4 page)

b) Relevant background to the project (UP TO 2 A4 pages) c) A Detailed plan of research; this should list each specific objective identified in (a) above

with a description of how it will be achieved experimentally

d) Justification of any resources requested e) Impact of the proposed research; this should identify who will benefit from the research,

how they will benefit and the methods or activities through which you will communicate and engage with those who benefit.

f) Any ethical considerations; this should identify any ethical permissions you may require (d-f UP to 1 A4 page)

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a) A summary of the aims and objectives of the project and the importance of the research being proposed (UP TO 1 A4 page)

Aims/objectives 1. To examine a range of candidate RNA aptamer molecules previously identified through in vitro

selection to HPV16 E6

2. To transfect DNA sequences encoding the chosen aptamers into a number of cell types including cells of; a more easily transfected cell line such as 293T cells, a cervical cancer cell line such as SiHa cells and a control cell line such as HaCaT cells

3. To determine if any of the RNA aptamer molecules expressed are capable of inducing apoptosis within cervical cancer cells i.e. the consequences of RNA aptamer(s) binding to E6

4. To help determine the course of future work with the aptamers

Importance of the research HPV16 infection causes roughly 50% of cervical cancers (Braaten and Laufer, 2008; Clifford et al., 2003), a disease that affects millions of women worldwide. Approximately 493,000 cases of cervical cancer are diagnosed and 233,000 deaths reported annually across the globe as a result of the disease (Parkin et al., 2005). Research in this field has the potential to significantly impact the lives of many women, along with the lives of individuals affected by other cancers resulting from HPV16 infection, such as those of the penis, vagina and head and neck carcinomas (Boulet et al., 2007; Gillison, 2004). This is because the aptamers under investigation may have applications as therapeutic and/or diagnostic agents in the future; in the treatment and/or identification of HPV16 infection that can cause cancers within a number of tissues.

This research may aid and encourage the development of aptamers to bind various proteins implicated in other diseases that lack suitable therapies such as Alzheimer’s disease, proteins of additional HPV strains, or, HPV16 viral proteins that are not as well characterised, such as oncoprotein E5. This could lead to developments in treating other diseases, other HPV infections (such as HPV18 also implicated in cervical cancer) and would allow the study of effects on HPV16 cervical cancer proliferation in the absence of a viral oncoprotein that is poorly understood, in comparison to E6 and E7 oncoproteins.

Additionally, scientists in the field of cervical cancer research would be able to build on the findings if they were promising, for instance, by studying the interactions of aptamers of interest in vivo. Future research may also involve expression of E6 and E7 aptamers of interest within the same cervical cancer cells, with the hope of inhibiting the actions of both major viral oncoproteins.

This research is therefore important to further knowledge in the field of cervical cancer research through the development of potential techniques for diagnosis and treatment of HPV16 infection. Findings would also apply to a number of different cancers resulting from HPV16 infection and techniques may be utilised for the diagnosis and treatment of other HPV infections, along with other forms of cancer and additional diseases with suitable proteins to target.

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b) Relevant background to the project (UP TO 2 A4 pages) Human Papillomaviruses Papillomaviruses were first described in 1907 (Ciuffo, 1907) and are a family of small, non- enveloped, double-stranded DNA viruses that are highly species specific. More than 150 types of human papillomaviruses (HPVs) exist, spanning five different genera (de Villiers et al., 2004). The alpha genus consists of around 40 different HPVs that infect the anogenital tract (Byg et al., 2012). These HPV infections represent the most common sexually transmitted viral infection (Markowitz et al., 2007) and can result in a range of abnormalities; from genital warts to cancers. HPVs of the alpha genus are classified as low-risk (LR) or high-risk (HR) according to the tendency towards malignant transformation of infected cells (Lorincz et al., 1992). LR types include HPV6 and 11 and HR types include HPV16, 18, 31, 33 and 45; these HPVs are associated with a number of epithelial cancers including those of the vagina, anus, vulva, penis, oropharynx, head, neck and mainly, the cervix (Blanchette and Branton, 2008; Boulet et al., 2007). Cervical cancers account for 12% of female malignancies corresponding to the second most prevalent cancer among women worldwide (Parkin et al., 2005), with around 500,000 cases diagnosed each year, one third of which are fatal (de Villiers, 1989). 99% of cases are the result of HR HPV infection (Boulet et al., 2007) and HPV16 is the most prevalent, responsible for over 50% of cervical cancers (Braaten and Laufer, 2008; Clifford et al., 2003). However, in most cases, HPV infections are transient and cleared by the immune system within 12-18 months (Richardson et al., 2003). It is thought that the major risk factor for malignancy is the persistent infection of HR HPVs (Markowitz et al., 2007), as this permits the accumulation of genetic abnormalities that allow transformation of cells and cancer development. For instance, HPV infections that lead to cervical cancer usually persist for decades.

Viral genome and life cycle The genomes of HPVs are circular, roughly 8kb in size, encode eight genes and consist of three main regions; an upstream long control region (LCR), an early region and a late region (see figure 1) (Burk et al., 2009; Ganguly and Parihar, 2009).

HPVs infect epithelial cells or keratinocytes within the basal layer of epithelium in the genital tract following wounding, where the virus synchronises its life cycle to the changes in differentiation that occur in host cells (Bodily and Laimins, 2010). Following entry and uncoating the viral genome is replicated episomally within the cell nucleus to roughly 100 copies (Moody and Laimins, 2010).

As host keratinocytes differentiate and leave the basal layer episomes are copied to 1000s within the cells and viruses are assembled (Bodily and Laimins, 2010). These virions are then released into the surrounding tissue as cells undergo desquamation.

Oncoproteins The cellular interactions of E5, E6 and E7 proteins promote tumourigenesis in persistent HPV16 infection. Each of these oncoproteins interferes with a number of signalling pathways. The mechanisms underlying the action of E5 are not fully elucidated, yet it is thought to modulate cell signalling to enhance that of growth factors such as epidermal growth factor (Bouvard et al., 1994) and prevent Fas- and TRAIL-mediated apoptosis (Kabsch and Alonso, 2002) to enhance immortalisation of cells by E6 and E7 and result in transformation and anchorage-dependent growth. The interactions of E6 and E7 are much more understood, and both are known to interact

Figure 1. The HPV16 circular genome. Transcription of six early genes; E1, E2, E4, E5, E6 and E7, and two late genes; L1 and L2 is regulated by the LCR (Fehrmann and Laimins, 2003). E1 and E2 are involved in initiation and regulation of viral replication, E4 and E5 are thought to be involved in late viral functions, E5, E6 and E7 are oncoproteins that ensure the maintenance of the viral genome, while, L1 and L2 proteins have structural roles, forming the icosahedral capsid of the virus (Bodily and Laimins, 2010).

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with a range of cellular proteins. Primarily, E6 binds with E6AP to the tumour suppressor protein p53 to result in its ubiquitination and proteosomal degradation (Scheffner et al., 1993; Scheffner et al., 1990). E6 is also known to interact with proteins involved in regulating transcription and DNA replication, apoptosis and immune evasion, epithelial differentiation, cell-cell adhesion, polarity and proliferation and DNA repair processes (Garnett and Duerksen-Hughes, 2006). The major interaction of E7 is binding to the tumour suppressor protein pRb to inhibit binding of E2F to pRb and allow cell cycle progression (Dyson et al., 1989). E7 binding to pRb can also result in ubiquitin- mediated degradation of the tumour suppressor protein (Fehrmann and Laimins, 2003). Additional E7 interactions take place with various proteins involved in cell cycle control, transcription regulation and other cellular functions (Garnett and Duerksen-Hughes, 2006).

E6 and E7 viral proteins therefore attenuate two major tumour suppressor proteins. Consequently, apoptosis and DNA repair pathways are repressed in infected cells, allowing survival of cells with damaged DNA as p53 is absent, while, replication and proliferation of cells is unregulated without the pRb protein. Ultimately, cells undergo proliferation, immortalisation and malignant transformation following expression and deregulation of E6 and E7 as viral genes integrate into the host genome (Boulet et al., 2007) and mutations accumulate. These oncoproteins have synergistic functions and both are required to induce cervical cancer formation in vivo (Howie et al., 2009). Treatment Although there are methods available to detect abnormalities that may lead to cervical cancer and prevent cervical cancer; the Pap smear and recently developed prophylactic vaccines to HPVs 16 and 18 respectively; treatment is currently limited to removal of lesions, through surgery or cryotherapy (D’Abramo and Archambault, 2011). There is therefore a need to develop novel therapies and targeting oncoproteins E5, E6 and E7 with small molecules such as RNA aptamers has great potential.

RNA aptamers RNA was first found to be involved in catalysis in the 1980s (Kruger et al., 1982). Subsequently, in 1990, three separate groups of researchers developed methods to select and isolate short RNA and DNA molecules in vitro, to bind specific targets (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990). The RNA molecules produced are short, single-stranded non- coding RNA aptamers that today are frequently created by automated SELEX (systemic evolution of ligands by exponential enrichment), based on those methods. SELEX involves rounds (usually 5-20) of identification and amplification of nucleotides that bind with high affinity to a chosen target (usually a protein) from libraries of up to 1015unique sequences (Blank and Blind, 2005; Burnett and Rossi, 2012). The synthetic aptamer molecules produced are usually 12-30 nucleotides in length and fold into precise, stable three dimensional structures that possess high affinity and specificity to the selected target and bind through complementary shape interactions (Bouchard et al., 2010; Ireson and Kelland, 2006).

Frequently referred to as ‘chemical antibodies’, aptamers are potent inhibitors of protein function, non-toxic and non-immunogenic, efficient and cost effective, can be selected against any type of molecule/molecular complex and easily produced and manipulated within the laboratory; for example, to increase biostability, modified bases can be added to prevent nuclease degradation (Blank and Blind, 2005). Aptamers are therefore advantageous over traditional antibodies.

Aptamers are highly versatile and can be applied to many fields such as therapeutics. The first RNA aptamer approved for clinical use was Macugen in 2004, to treat age-related macular degeneration (Bunka et al., 2010; Gragoudas et al., 2004). This success has fuelled the development of many more therapeutic aptamers, and a number are currently in clinical trials.

RNA aptamers have previously been selected against HPV16 E7, with promising results including loss of E7 from and apoptosis of cervical cancer cells (Nicol et al., 2011; Nicol et al., in press). E6 will now be targeted in the same way in the hope of blocking the protein. This would prevent numerous cellular interactions including binding to and degradation of p53, and ultimately, cervical cancer progression. Blocking E6 would also allow induction of apoptosis in HPV16 infected cells.

c) A Detailed plan of research; this should list each specific objective identified in (a) above with a description of how it will be achieved experimentally

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1) To examine a range of candidate RNA aptamer molecules previously identified through in vitro selection to HPV16 E6

The aptamers under investigation will be chosen from a pool of aptamers previously identified via SELEX to the HPV16 E6 oncoprotein and 10-12 will be studied in human cells.

2) To transfect DNA sequences encoding the chosen aptamers into a number of cell types including cells of; a more easily transfected cell line such as 293T cells, a cervical cancer cell line such as SiHa cells and a control cell line such as HaCaT cells

293T cells are derived from a human embryonic kidney cell line and are widely utilised and relatively easy to culture and transfect. Use of this cell line would allow assessment of transfection efficiency and could indicate the half lives of aptamers prior to use of other mammalian cells. These cells would also act as a control for cell type, helping to ensure any observations following aptamer expression are specific to cervical cancer cells. SiHa cells are from a human cervical carcinoma cell line resulting from HPV16 infection, possessing chromosomal insertion of HPV16 sequences and therefore expressing E6 and E7 oncoproteins, while HaCaT cells are from an immortal human keratinocyte cell line and represent normal epithelial cells (i.e. found in the cervix and infected by HPV16 in cervical cancer) expressing p53 and pRb. As these cells do not express E6 or E7, they can act as a control to confirm any findings are specific to cervical cancer cells and do not apply to normal epithelial cells found in the cervix.

These mammalian cell lines will be cultured appropriately; in media such as Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with components including foetal calf serum (FCS) and antibiotics such as penicillin and streptomycin. This would provide optimum growth conditions for the cells and prevent contamination of cultures.

As cells are being cultured, cloning of RNA aptamer cDNA sequences will take place into mammalian expression vectors such as pSUPER (Oligoengine). This plasmid vector possesses a polymerase III HI-RNA gene promoter, unique BgIII and HindIII/Xhol restriction sites, ampicillin resistance and is available with puromycin or neomycin resistance genes (see figure 2). pSUPER therefore initiates transcription of mammalian genes and increases the ease of cloning and selection of cells possessing the recombinant vector produced. Other suitable vectors include pSIREN and pSILENCER. pSUPER would however be preferred and has been utilised to successfully introduce shRNA (Chen et al., 2013; Zhang et al., 2012), siRNA (Niu et al., 2012) and microRNAs (Li et al., 2012) into mammalian cells.

Prior to insertion into the vector, DNA sequences encoding RNA aptamers may be amplified using PCR, and restriction sites incorporated flanking the DNA, such as BglII and HindIII sites recommended for use with pSUPER. Both vector and aptamer sequences can then be cut with these restriction enzymes to yield complementary, cohesive ends, and ligated to form a

Figure 2. pSUPER vector possessing neomycin resistance (Oligoengine, 2004).

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recombinant vector. Transformation of competent bacterial cells such as Escherichia coli DH5α cells with recombinant vector would allow determination of transformation efficiency in comparison to positive (vector without insert) and negative (water only) controls and selection of transformed bacterial cells containing the desired recombinant plasmid vector with the antibiotic ampicillin. Presence of the insert can then be confirmed using PCR and sequencing.

Once the presence of insert has been confirmed in the vector, transfection of mammalian cells using a method such as electroporation or lipofection can take place, including transfection of a positive control vector and a negative control/mock transfection. Stable cell selection will follow, using neomycin or puromycin depending on the variant of pSUPER utilised, with two non- transfected controls; one that is subjected to antibiotic selection (when all these cells are killed selection is complete) and one that is not (to control for cell viability).

Following transfection of aptamers into the cells, expression levels may be determined using a method such as quantitative RT-PCR, with nucleic acid primers complementary to regions within the aptamer sequences.

3) To determine if any of the aptamer molecules expressed are capable of inducing apoptosis within cervical cancer cells i.e. the consequences of RNA aptamer(s) binding to E6 If one or more of the aptamers selected for study is able to bind E6 and prevent its binding to and degradation of p53, p53 would then be able to induce apoptosis in the cervical cancer cells. In order to determine whether or not cells are undergoing apoptosis, a number of assays could be carried out. Cells may be stained with propidium iodide (PI), an intercalating agent and fluorescent molecule that stains DNA within cells and is generally only taken up by cells that are dying. Analysis of stained cells can be achieved using flow cytometry. PI staining indicates cell death, however, alone is unable to distinguish between apoptotic and necrotic cells. Staining with annexin V and PI prior to flow cytometric analysis would help to confirm cells undergoing apoptosis, as annexin V detects the externalisation of phosphatidylserine protein.

Transfection of sequences encoding a control aptamer such as staurosporine, or a randomly synthesised aptamer into cells and study of apoptosis would ensure that findings were not the result of the vector used, but of the aptamer(s).

4) To help determine the course of future work with the aptamers Future work would then involve studying the binding activities of any RNA aptamers of interest identified from the apoptosis assays to E6 in vitro and resulting affects on p53 binding and E6 levels, along with the nature of any interactions and results on cell proliferation. Decreases of both the amount of p53 bound to E6 and levels of E6 protein would be expected in the presence of successful aptamers.

Aptamer binding assays may be undertaken with a fusion protein such as GST-E6 following expression and purification in cells such as BL21 (DE3) cells. RNA immunoprecipitation experiments would allow study of aptamer and E6 interactions, co-immunoprecipitation studies would help determine the E6 and p53 interactions before and after transfection and affinity chromatography such as GST pull-down assays with immunoblotting would help characterise any interactions. Immunoblotting could also determine E6 levels and cervical cancer cell proliferation assays may be carried out using a system such as xCELLigence (Roche, UK).

In addition, interactions should be studied within other cervical carcinoma cell lines such as; C33A cells that do not express any HPV DNA sequences (control), CaSki cells that express HPV16 DNA sequences (experimental) and HeLa cells that express HPV18 DNA sequences (control).

d) Justification of any resources requested The proposed investigation will be undertaken at the University of Leeds, in collaboration with Dr Nicola Stonehouse who possesses both wide ranging knowledge and experience of working with

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RNA aptamers and various viruses including HPV16 (Nicol et al., 2011; Nicol et al., in press; Wetherill et al., 2012).

The resources needed include; cells lines, culture medium, vector, restriction enzymes, and reagents required to express RNA aptamers in cervical cancer cells and study the ability of cells to induce apoptosis. All of the equipment required is accessible within the laboratory.

e) Impact of the proposed research; this should identify who will benefit from the research, how they will benefit and the methods or activities through which you will communicate and engage with those who benefit.

The major beneficiary of this research is women worldwide currently diagnosed with cervical cancer resulting from HPV16 infection, along with individuals with other forms of cancer associated with HPV16 infection, and those infected in the future, as these individuals may be able to receive more effective diagnosis and treatment. Other scientists within the field of cervical cancer research resulting from HPV16 and other high-risk HPV types such as HPV18 may benefit from the research if results are promising through the methods utilised and a boost in morale. These researchers may also want to further their own work by building on the findings, while scientists in the fields of other forms of cancer and disease research may benefit, again, from the approach utilised such as the use of aptamers.

Findings could therefore have a greater impact than cervical cancer alone as the techniques could be applied to other cancers associated with HPV16 and other HPV infections, and potentially, additional forms of cancer and diseases that may or may not involve viral infection, as aptamers can be applied to block almost any target. Thus, this research has the potential to impact women worldwide, the general public and the scientific community.

Any significant developments will be communicated to the scientific community by attending conferences both in the United Kingdom and abroad to present and discuss findings, publishing in peer-reviewed journals and also in an electronic journal to allow easy access to the data. Findings could be communicated to patients by making the information available on educational websites such as MEDLINEplus. Finally, the general public may be notified through scientific media such as magazines, and public media via press releases together with the University of Leeds press offices.

f) Any ethical considerations; this should identify any ethical permissions you may require (d-f UP to 1 A4 page)

The planned experimental procedure involves the use of various cell lines, cloning of sequences encoding RNA aptamers, transfection into cells and analysis via apoptosis assays. Ethical permissions are therefore not required.

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