An HRE-binding Py-Im polyamide impairs hypoxic signaling in tumors

An HRE-binding Py-Im polyamide impairs hypoxic signaling in

tumors

Jerzy O. Szablowski

Jevgenij A. Raskatov

1,*

, and

Peter B. Dervan

a,1

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

CA 91125

Abstract

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ

fibrosis, age-related macular degeneration and cancer. HIF-1, a transcription factor central to the

hypoxic gene expression, mediates multiple processes including neovascularization, cancer

metastasis and cell survival. Py-Im polyamide 1 has been shown to inhibit HIF-1-mediated gene

expression in cell culture but its activity

in vivo

was unknown. This study reports activity of

polyamide 1 in subcutaneous tumors capable of mounting a hypoxic response and showing

neovascularization. We show that 1 distributes into subcutaneous tumor xenografts and normal

tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation

of new tumor blood vessels and suppresses tumor growth. Tumors treated with 1 show no increase

in HIF-1a and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased

apoptosis in HIF-1a positive regions and the increased proximity of necrotic regions to

vasculature. Overall, these results show that a molecule designed to block the transcriptional

activity of HIF-1 has potent anti-tumor activity

in vivo

, consistent with partial inhibition of the

tumor hypoxic response.

Keywords

hypoxic signaling; angiogenesis; Py-Im polyamide; phenotypic mechanism; gene expression

Introduction

Oxygen sensing is involved in a range of natural physiological processes such as embryonal

development, wound healing and immune response (

). However, its dysregulation can

contribute to pathogenesis of multiple diseases including fibrosis, erythrocythemia, heart

disease and cancer - a group of diseases leading to nearly 600,000 deaths every year, in the

United States alone (

). While new cancer treatments are being developed, many of the

cancer therapies are hampered by presence of low levels of oxygen in tumors (

), known as

tumor hypoxia, regulated mainly by Hypoxia Inducible Factors (HIFs) (

). Among them -

To whom correspondence should be addressed: ; Email: dervan@caltech.edu, California Institute of Technology, Division of

Chemistry and Chemical Engineering, MC 164-30, Pasadena, California 91125.

Current address: Jevgenij A. Raskatov, Dept. of Chemistry and Biochemistry, 1156 High St., University of California Santa Cruz, CA

95064

Conflicts of interest

: The authors have no conflicts of interest to declare.

HHS Public Access

Author manuscript

Mol Cancer Ther

. Author manuscript; available in PMC 2017 April 01.

Published in final edited form as:

Mol Cancer Ther

. 2016 April ; 15(4): 608–617. doi:10.1158/1535-7163.MCT-15-0719.

Author Manuscript

HIF-1 (

) - is a transcription factor that is often is associated with poorer patient survival (

HIF-1 orchestrates numerous aspects of cancer progression - tumor angiogenesis, cell

survival in hypoxia and metastasis (

). Most inhibitors affect HIF-1 signaling indirectly,

by targeting other proteins, including Topoisomerase I, mammalian target of rapamycin

(mTOR) or microtubules (

). Established therapeutic strategies focus on modulating

HIF-1 signaling by altering the HIF-1 protein levels (

), its dimerization and protein

interactions (

–

), or its DNA binding and transcriptional activity (

–

One method to inhibit transcriptional activity of HIF-1 is to displace it from its DNA-

binding site – HIF-1 responsive elements (HREs), for example by a molecule shown in this

report (compound

. Fig. 1A) (

). Compound

is a member of a class of DNA-binding

small molecules – Pyrrole-Imidazole (Py-Im) polyamides - which can be programmed to

recognize a broad repertoire of sequences with affinities and specificities comparable to

those of transcription factors (

). We reported that polyamides can modulate gene

expression driven by many transcription factors in tissue culture (

–

Experiments focused on HIF-1-DNA binding inhibition demonstrated the ability of

displace the HIF-1 complex from DNA

in vitro

(

), and by reducing HIF-1a occupancy at

selected HREs in a common tissue culture model of hypoxic response - U251 cells as

represented in Fig. 1B (

). When U251 cells were treated with Deferoxamine, an iron

chelator and HIF-1 activator (

was capable of inhibiting 23% of the induced genes,

establishing polyamide

as a partial inhibitor of HIF-1 transcriptional activity (

). The

concentration used was 1 μM, well below what was measured in the tumor tissues

investigated in this study (2–3.5 μM). The overall gene expression changes included many

important proangiogenic factors, such as

VEGFA

FLT1

Endothelin-1

, but also other

factors. In light of the recent developments in cancer systems biology (

) the broad

effects of Py-Im polyamides on gene expression

(

–

) led us to posit that their

effects may arise due to affecting multiple targets. Recent discoveries show that drugs

commonly act upon multiple molecular entities (

) and this feature could be beneficial

in cancer treatment thanks to decreased likelihood of developing drug resistance (

However the potential for toxicity due to off target effects is a point of concern. Our

mechanistic investigations expanded beyond the transcription factor:DNA interface and

found that Py-Im polyamides can induce degradation of the large subunit of RNA

polymerase II, activation of the P53 stress response without concurrent DNA damage (

)

and induction of DNA replication stress (

We decided to establish whether the pattern of gene expression caused by administration of a

Py-Im polyamide binding DNA sequence found in HREs (5

′

-WTWCG-3

′

) could result in

measurable and useful anti-tumor effects

in vivo

. Our experiments focused on suppression of

the phenotypes typical for tumor hypoxia, such as tumor vascularization, hypoxic gene

expression or cancer cell survival (

). We chose a subcutaneous tumor model, due to its

hypoxic nature (

) and reliability of these measurements. The cell lines chosen for

engraftment have been evaluated extensively in xenograft models of cancer (

) and

hypoxic gene expression in one of them (U251) can be regulated by

(

), making it a good

choice for evaluating

in vivo

mechanism of action of

. The second cell line, GBM39 (

was derived from the same site as U251 cells (brain), but was maintained as subcutaneous

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xenografts and expanded in serum-free conditions as spheroids. The serum-free treatment

maintains genetic and histologic variability of human tumors and was thus our choice to test

generality of mechanism of action of polyamide

(

). Our previous

in vivo

investigations

demonstrated the bioavailability of various Py-Im polyamides upon intravenous (

intraperitoneal and subcutaneous administration (

). Subsequent

in vivo

xenograft studies

established that polyamides could accumulate in subcutaneously grafted tumors (

modulate tumor gene expression (

) and inhibit growth of prostate cancer xenografts

(

). The distinct profile of gene expression modulation, favorable pharmacokinetics and

ability to modulate all of the tested aspects of hypoxic response by

, could establish it as an

interesting candidate for treatment of cancer and other hypoxia-related diseases. In the

current study we established the ability of Py-Im polyamide 1 to interfere with hypoxic

response, regulate gene expression in tumors and to inhibit tumor growth and angiogenesis.

Materials and methods

Cell Maintenance

U251 cell line was a kind gift of Dr. Giovanni Mellilo (NCI) in 2005. GBM39 cells were a

gift of Dr. David Akhavan (UCLA) in 2013, originally obtained from Prof. David James

(UCSF). The cell lines were authenticated using StemElite ID assay (Laragen). U251 cells

were maintained in RPMI-1640 medium supplemented with 10% FBS. GBM39 cells were

cultured in suspension in

F12/DMEM medium supplemented with b27, EGF (20 ng/ml) ,

FGF2 (20ng/ml), and Glutamax, heparin (50 μg/ml),

with growth factors replaced every two

days. After 25 passages GBM39 were reestablished as subcutaneous xenografts (NSG mice).

The tumor was disintegrated, digested in 10% accutase (3 h), cells resuspended by pipetting

and then cultured as described above. Human umbilical vein endothelial cells (HUVECs)

were cultured in 200 PRF medium (Gibco) supplemented with Low Serum Growth

Supplement (LSGS, Invitrogen). Media was exchanged every 48 h and cells passaged

weekly.

Tube formation assay

HUVECs were plated at 2×10

cells (75cm flask). After 36 hours, compound

(5 μM) was

added for 72 h. Twelve-well plates were coated with 100 μl/well Geltrex (Invitrogen) and

allowed to solidify at 37°C for 60 min. Cells were trypsinized and resuspended in 200 PRF

medium at 2×10

−1

, and plated in the 12-well plates at 400 μL per well. After 6–12

hours the wells were imaged on an inverted microscope by selecting 4 random fields of

view.

Pharmacokinetic and toxicity experiments

Animal experiments were carried out according to approved Institutional Animal Care and

Use Committee protocols at the California Institute of Technology (Pasadena, CA). PK

studies were done as previously described (

). Serum for analysis of toxicity markers

(IDEXX) was obtained by RO collection and centrifugation (2000g, 15 min) in Serum

Separator Tubes (BD Biosciences). The radioquantitation of

was performed as described

elsewhere (

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Immunohistochemistry and histologic analysis

Tissue processing was conducted at the Tissue Processing Core Laboratory (TPCL) at

UCLA, using 5 μm paraffin sections. Tumor microvessels were visualized using anti-mouse

CD31 staining using SC-1506 antibody (SCBT), apoptosis was assayed for using anti-

human Cleaved-Caspase-3 (CC3) antibody (9664, CellSignal) and HIF-1a using CME 349A

(Biocare Medical). At least two tissue sections per slide were used for each experiment.

Slides were scanned using an Aperio ScanScope AT (Aperio) and positive pixel

measurements were done using Imagescope software.

Perinecrotic area was defined as a field of view at 20× magnification that contained 50%

necrosis and 50% adjacent, non-necrotic area, with edge of necrosis in the middle of the

screen. Number of histologic measurements for each tissue section were chosen to limit

variability of the final average value to <10%, per tumor, or until all available data in the

section was counted. Ki-67 measurements were done using Immunoratio software at 20×

magnification, with n=24 per tumor section. Nuclear density (and by proxy – necrosis) was

assessed by automatic nuclei counting using Image-based Tool for Counting Nuclei (ITCN),

16 fields of view were analyzed per tumor section.

At least 6 (CC3), 9 (CD31), or 3 (TUNEL, GBM39), 5 (TUNEL, U251), or 8 (HIF) fields of

view per tumor chosen randomly prior to analysis, at either 20× (CC3 staining, CD31

staining for U251, GBM39, Schedules A and B, TUNEL, HIF) or 40× magnification (CD31

staining for GBM39,

Schedule C

, due to higher CD31 positive vessel counts). For analysis

of frequency of apoptotic blood vessels (CC3/CD31 double staining), we counted enough

high power fields (20x) containing CD31 positive vessels, to assess at least 600 microvessels

per condition.

Perinecrotic MVD was measured by first delineating nuclei on the edge of necrosis, and then

offsetting the obtained path by an equivalent of 50 μm-350 μm in Illustrator CS5 (Adobe).

The MVD was then counted manually for each 50 μm-zone and area integrated in Photoshop

CS5 (Adobe). At least 40 measurements per tumor were obtained. Measurements of the

nearest blood vessel-necrosis distance were done manually using Imagescope at 40×

magnification.

Xenograft experiments

Experiments were carried out in 8–12 week old male NSG mice (NOD.Cg-Prkdcscid

Il2rgtm1Wjl/SzJ, JAX). Cells were injected s.c. into a flank area (2.5×10

of U251 and

7.5×10

of GBM39 cells). U251 cells were administered in RPMI-1640 medium and

GBM39 cells in 50% matrigel/culture medium. Before treatment, each vehicle-treated

control xenograft was paired with a xenograft of the same initial volume to be treated with

Compound was administered s.c. interscapularly in 20% DMSO/PBS. Mice were weighted

weekly and euthanized if 15% weight loss, or more was observed.

Quantitative Real Time-Reverse Transcription-PCR assay

Primers (Supplementary Table 1) were designed using Harvard Primer bank (

). The total

mRNA in tumors was processed as in our previous studies (

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RNA-sequencing analysis

Cells for gene expression analysis were plated in 6-well plates at 5–10 × 10

cells per well

and incubated in RPMI-1640 medium with 10% FBS until ~50% confluence (24–48 h).

Afterward, medium was exchanged with the growth medium supplemented with

(1 μM).

After 48 hours, either 300 μM of DFO, or PBS was supplemented, cells were incubated for

an additional 16 hours. The RNA was then harvested using an RNEasy Kit (Qiagen) and

treated with a Riboguard RNAse inhibitor and TurboDNA Free DNAse (Ambion), according

to manufacturers' instructions. RNA-Seq libraries were prepared using standard Illumina

reagents and protocols. Single read sequencing with the read length of 50 nucleotides was

conducted on the Illumina HiSeq2000 sequencer, producing 35 to 40 million reads per

library, with a total of three biological replicates per sample. Sequencing data were mapped

against the combined human (hg19) transcriptome, using the Tophat2 program package

2.0.13 (

) and the GRCh37 annotation. Differential gene expression was calculated with

the DEseq2 module (

). Clustering analysis was performed using Cluster 3.0 software (

Statistical analysis

Each sample was analyzed using a two-tailed, student’s t-test, assuming normality and

unequal variance. P-value of 0.05 or less was considered statistically significant.

Results

Activity of Py-Im polyamide 1 depends on the HIF-1a activation

Our previous results showed that

can act as an inhibitor of HIF-1-driven gene expression

(

) in U251 cells. However, we did not determine activity of

in cells without HIF-1a

induction. Tumors contain both hypoxic and normoxic regions and thus it is important to

establish how

acts in cells with and without induced HIF-1a. To address this question,

U251 cells were first treated with

(1 μM, 48 h) or PBS (vehicle). Half of the samples were

then dosed with a HIF-1a inducer Deferoxamine (DFO, 300 μM, 16 h), while the other half

with PBS (Supplementary Figure 1A). Their mRNA was then analyzed by RNA-sequencing.

Gene expression differences for each sample were obtained by normalizing a polyamide-

treated sample with the appropriate non-treated control – cells treated with PBS and

were

normalized to cells treated with PBS only (PBS vs

), and cells treated with both

and DFO

to cells treated with DFO (DFO vs DFO+

). When

was dosed into cells treated with DFO,

we observed it downregulated 744 genes. When

was dosed into cells dosed with PBS, a

different set of genes was regulated. In fact only 101 out of 723 regulated genes were

identical for both conditions (14%, Fig. 1C, D). The upregulated genes also varied between

the two groups. Cells treated with

and DFO showed upregulation of 316 genes, while the

same cells treated with

and PBS – 1514 genes. Few genes were in common between the

two groups (6% for DFO, 1.3% for PBS, Fig. 1C, D). Overall, 17% DFO-induced genes

were downregulated by

at least 2-fold (p < 0.05). Gene clustering analysis suggested

acted to reverse effects of DFO (Supplementary Figure 1B–C). Fewer genes were affected

by compound

at the same dose (Supplementary Figure 1D). Interestingly, we found that

among the genes that were changed significantly both by

(in DFO-induced cells) and by

DFO, the Py-Im polyamide

inhibited DFO-induced gene expression in 96% of the genes

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(2557 out of 2661 genes, Supplementary Figure 1E). This indicates that gene expression

changes caused by DFO, are either unaffected, or reversed by

, but rarely, if ever,

exacerbated by treatment with

Py-Im polyamide 1 shows favorable preliminary pharmacokinetics and tissue distribution

at tolerable doses

In order to evaluate bioavailability of

in vivo

, we injected it subcutaneously (s.c.) at 6.8

mg/kg into C57BL6 mice. Compound

reached a serum concentration of 11.3±1.6 μM

within 1.5 h and 7.8±0.4 μM at 4 h post-injection (n=4, Fig. 2A). The radiolabeled analogue

, Py-Im polyamide

, was injected intraperitoneally (IP) to quantitate whole-organ

compound concentrations (n=6, Supplementary Figure 2A, B) into immunocompromised

NSG mice bearing GBM39 tumors. After 24 h, concentrations of

were measured in

GBM39 xenografts (2.0 μM), host’s kidney (6.3 μM), liver (4.2 μM) and lung (3.1 μM;

Supplementary Figure 2A). Following 3 injections (6.8 mg/kg s.c., every other day × 3,

harvest 24h post last injection), all tested tissues showed compound accumulation

(Supplementary Figure 2B). The same administration of

(FITC-conjugate of

) in NSG

mice resulted in readily detectable nuclear staining in tumor cells and tested tissue sections

(Supplementary Figure 2C–E). The nuclear staining was also present when tested in unfixed

cells in tissue culture setting (Fig. 2B). A single-injection toxicity test showed that

did not

affect animal weight, or levels of serum toxicity markers (ALT, AST, TBIL, BUN,

Creatinine) at concentrations up to 10 mg/kg (Supplementary Figure 3A, B). Taken together,

these results supported evaluation of anti-tumor effects of

in vivo

Polyamide 1 suppresses tumor growth in subcutaneous xenografts

To test whether previously established partial inhibition of HIF-1-driven gene expression by

(

) would result in decrease in tumor growth, we engrafted U251 and GBM39 cells s.c.

into NOD/SCID-

(NSG) mice. We established three dosing regiments (Supplementary

Figure 4A) for

–

Schedule A

, consisting of three s.c. injections every other day at

maximum tolerated dose (6.8 mg/kg, 8 days) and

Schedules B and C

, with lower dose

(4.5mg/kg, 3 inj./week) designed for prolonged treatment with low weight loss (<10%) over

4 weeks (U251 tumors) or 6 weeks (GBM39 tumors). The S

chedule A

was designed to elicit

maximum response over a short period of time at a dose tolerable to animals. The

Schedules

and

were introduced to assess if the compound

could retain its activity at a dose that

could be tolerated over longer periods of time. In all cases, the tumors were harvested 72 h

after last injection, due to our previous research, indicating that Py-Im polyamide-induced

apoptosis occurs between 48–72 hours after compound administration in tissue culture

studies (

Mice bearing U251 tumors were subjected to treatment with

(

Schedule A

) which resulted

in a reduction in median tumor mass (1.8-fold, p<0.013; n=7, 8 for vehicle and treated

groups; Fig. 2C) compared to vehicle. Similarly, prolonged treatment (

Schedule B

) resulted

in 1.9-fold lower median U251 tumor mass (p<0.0125; n=10 per condition; Fig. 2D).

Consistent results were obtained with a primary glioma cell line (GBM39) xenograft.

Treatment according to

Schedule A

resulted in 1.8-fold lower median mass (p<0.016; n=11

per condition; Supplementary Figure 4B) and prolonged treatment (

Schedule C

) in 1.6-fold

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reduction (p<0.041; n=6 per condition; Supplementary Figure 4C). The treatments resulted

in minor mouse weight loss; the effects were less severe (<5% average w.l. throughout the

experiment) for schedules

and

(Supplementary Figure 5).

Polyamide 1 reduces microvessels density in xenografts, without inducing endothelial

apoptosis

Hypoxic signaling is a major driver for angiogenesis and its inhibition leads to decrease in

microvessel density (MVD) (

). We measured MVD using anti-mouse CD31

immunostaining of tumor sections. We observed a significant reduction of MVD in all tested

scenarios. For U251 tumors, with mice subjected to

Schedule A,

we observed a 1.4-fold

median reduction of MVD (p<0.014; n=7, 8 for vehicle and treated groups; Fig. 2E) and 1.7-

fold median decrease for

Schedule B

(p<0.05; n=10 per condition; Fig. 2F). For GBM39

tumors, both treatment schedules (

and

) led to 1.4-fold reduction in MVD (p <0.01;

n=11 and n=6 for Schedules

and

; Supplementary Figure 6A–B).

The decrease in MVD could be caused by direct effect of

on the endothelium, for example,

rendering it either apoptotic, or unable to form blood vessels. We evaluated endothelial

apoptosis by double-staining of mouse-specific CD31 and Cleaved Caspase-3 (CC3) in

GBM39 tumors (

Schedule C

; n=5 tumors, approximately 600 microvessels per condition;

Fig. 2G). We found no significant endothelial apoptosis in either of the treatment groups. To

measure angiogenic functionality of endothelium we used

in vitro

matrigel tube formation

assay with Human Umbilical Cord Endothelial Cells (HUVEC), revealing

(5 μM, 48 h)

had no effect on tube formation (n=3, Fig. 2H).

Antiangiogenic effects of 1 are associated with inhibition of hypoxic response

Decrease in MVD, without endothelial apoptosis or dysfunction, suggests

could interfere

with the hypoxic response (

). To further test this hypothesis we evaluated other aspects

affected by hypoxic response, such as tumor cell proliferation (

), apoptosis in HIF-1

positive, perinecrotic areas (

), nuclear HIF-1a protein accumulation and cell survival in

areas distant from blood vessels (

). To analyze this, we divided the tumor sections into

three areas: necrotic, non-necrotic and perinecrotic (Fig. 3A). The non-necrotic areas contain

nucleated cells with intact cell membranes, while necrotic areas are show loss of nuclei and

fragmentation of cells. The perinecrotic area represents field of view (20× magnification)

that contains 50% of cells containing nuclei and 50% of necrotic areas.

Antiangiogenic therapy increases hypoxia in tumors and often leads to decreased

proliferation (

). Upon treatment with

(

Schedule B

), we observed a 1.2-fold decrease

(P<0.05; n=10 per condition; Fig. 3B) in proliferation marker (Ki-67) in non-necrotic areas

of the U251 tumors.

Another effect of induction of hypoxic response is accumulation of HIF-1a (

). However,

treatment with

did not lead to increase in levels of HIF-1a in either U251 (Fig. 3C, D) or

GBM39 (Supplementary Figure 7A, B) tumors. Lack of HIF-1a accumulation is unlikely to

be caused by its increased degradation, as

does not affect HIF-1a levels in tissue culture

(Supplementary Figure 7C). Overall, we observed a spatial distribution of HIF-1a in U251

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tumor sections – perinecrotic areas showed higher HIF-1a levels compared to non-necrotic

areas (1.8-fold average increase for

, p<0.001; n=10 per condition; Fig. 3C, D).

Induction of hypoxic signaling can lead to apoptosis resistance in cancer cells (

However, treatment with

increased apoptosis significantly in perinecrotic, HIF-1a positive,

areas (CC3 staining, 1.8-fold, p<0.0012; n=10 per condition; Fig. 3E;). The apoptosis was

absent in non-necrotic regions of U251 tumors regardless of the treatment (Fig. 3F),

suggesting

is toxic specifically to hypoxic cells. Similarly, TUNEL staining revealed

convergent results – no increase in apoptotic cells in non-necrotic regions, and 1.7-fold

(p<0.009, n=10 per condition) increase in apoptosis in perinecrotic tumor regions upon

treatment with

(Supplementary Figure 7D–E). The GBM39 tumors harvested before they

developed necrosis also revealed no induction of apoptosis by

(Supplementary Figure 7F).

Hypoxic signaling mediates cell survival in areas with low oxygen pressures. With

increasing distance to blood vessels, tumor necrosis appears as a result of the death of cells

with inadequate oxygen supply (

). The oxygen pressure decreases as the function of

distance from blood vessels, and typically does not reach beyond approximately 200 μm.

Cells adapt to low oxygen pressures by activating hypoxic signaling, and once oxygen

pressures drop below a critical value, cell necrosis occurs (

). The cells that succeed to

survive at distances comparable to 200 μm from blood vessels, can thus be deemed as having

a functional hypoxic response. Impairment of hypoxic signaling will result in necrosis

occurring at higher oxygen pressures – at shorter distances from blood vessels. Therefore to

measure if

decreases ability of cells to adapt to low oxygen pressures, we measured MVD

as a function of distance from the edge of necrotic areas. We found the expected lack of

microvessels near necrotic edges (<100 μm). In tumors derived from animals treated with

(U251,

Schedule B

) necrotic edge appeared closer to the blood vessels. The median

distances between the edge of necrosis and the nearest blood vessels, were significantly

shorter for both U251 and GBM39 tumors treated with

, as compared to vehicle-treated

controls (p<0.01, Supplementary Figure 7G). Similarly, at short distances around necrosis

the mean MVD appeared higher for tumors treated with

suggesting treated tumor cells

require higher level of nutrients and oxygen for survival (3.3-fold at 150 μm and 2.3-fold for

200 μm distance from the necrotic edge, p<0.02 and p<0.01; Fig. 4A, B).

Lower MVD and lower cell survival at a distance from blood vessels, should lead to an

increase in necrotic area. However, the complex pattern of necrosis (micronecroses) in U251

tumors made it difficult to quantify it by a simple delineation. Instead, we decided to

automatically count nuclei in the whole tumor section and found lower nuclear density in

tumors treated with

, (10% fewer nuclei, p<0.03, Supplementary Figure 8A). The further

evidence of necrosis induction by

, was present after short-term treatment with

GBM39 tumors (

Schedule A

) where transient accumulation of HIF-1a (Supplementary

Figure 8B–D) led to presence of necrosis (Supplementary Figure 8E), specifically localized

to HIF-1a positive areas that were distant from microvessels (Supplementary Figure 8E, F).

Overall, compound

decreases proliferation and nuclear density, selectively induces

apoptosis in perinecrotic, HIF-1a positive areas, and causes necrotic areas to appear in

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proximity of blood vessels despite the presence of a blood supply, but treatment does not

result in long-term HIF-1a accumulation.

Py-Im polyamide 1 reduces expression of proangiogenic and prometastatic factors

in vivo

A common adverse effect of antiangiogenic treatment is upregulation of proangiogenic and

prometastatic gene expression which renders the antiangiogenic therapies less effective and

is often a result of hypoxic signaling (

). We decided to test if

affected mRNA expression

of such factors

in vivo

. NSG mice bearing U251 tumors were treated with

according to

Schedule D

(6.8 mg/kg, 2 inj., on days 1 and 3; tumors harvested on day 5; SI

Supplementary Figure 4A). Out of 10 tested proangiogenic factors, four transcripts had

lower relative expression after treatment with

(Fig. 5A), and one (

VEGFA

) was

upregulated (1.4-fold) in both mRNA (Fig. 5B) and serum protein levels (Fig. 5C).

Downregulation of mRNA expression was also apparent in the panel of prometastatic genes.

Overall, 5 out of 6 tested transcripts were downregulated in the group treated with

(Fig.

5D). The factors significantly affected by

, included:

FLT1, NLGN1, HOXB9, NEDD9,

MMP2

and

MMP14

. Interestingly, the mouse receptor of Angiopoietin,

Tek,

was also

downregulated. Results for all tested genes can be found in Supplementary Table 2.

Discussion

Regulation of hypoxic signaling is central to maintaining balance between health and

disease. Its principal regulator - HIF-1 - is essential for tumor initiation and progression,

e.g.

vascularization, cell survival and metastasis (

). Inhibition of HIF-1 activity has

suppressed tumor progression and reduced cancer resistance to available therapies (reviewed

in (

)). Our group has previously reported on the function of an HRE-binding Py-Im

polyamide

as a partial inhibitor of HIF-1 dependent transcription in tissue culture (

However, the

in vivo

activity and mechanism of

remained to be explored. We used Py-Im

polyamide

(Fig. 1) to show its activity is dependent on HIF-1a activation (Fig. 1), that it

inhibits tumor growth, angiogenesis (Fig. 2) and the tested aspects of hypoxic response

vivo

(Figs. 3 and 4), including inhibition of tumor-derived mRNA expression of

proangiogenic and prometastatic factors that are often upregulated in hypoxic conditions

(Fig. 5). We found that the effects of

are consistent with what would be expected in case of

a partial inhibition of hypoxic response (Fig. 6).

Polyamide tissue distribution and preliminary pharmacokinetics

Subcutaneous administration revealed

distributes into serum within 1.5 h post-injection,

with 31% drop in concentration 2.5 h later, indicating multi-hour long half-life. Even though

had favorable pharmacokinetics, its activity

in vivo

is likely dependent on the target tissue

concentration. After three intraperitoneal injections (Schedule

), the radioactive analog

reached a concentration of 3.5 μM in GBM39 tumor and higher levels in other tissues.

Concentrations attained for

in all tested tissues were thus higher than used in our tissue

culture studies (

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Effects on tumor growth and vascularization

In the present study, inhibition of tumor growth and reduction in MVD was observed in two

different cell types in response to treatment with

. The extent of these effects was

comparable to ones exerted by Bevacizumab (

) in xenografts derived from U251 (

) and

GBM39 cells (

). Investigation of possible mechanisms of antiangiogenic action showed

lack of apoptosis in blood vessel lining, and no measurable influence on tube formation on

matrigel. Lack of direct effects of

on endothelial cells suggests blood vessel recruitment

might be impaired. One possible explanation is inhibition of expression of tumor-associated

proangiogenic factors by

. However, systemic concentration of those proteins could also be

affected.

Effects on apoptosis, proliferation and HIF-1a levels

Compound

induces apoptosis selectively, in HIF-1a-positive, perinecrotic areas. This

suggests

sensitizes cancer cells to hypoxia, which could occur by hypoxic response

inhibition (

). Our RNA-sequencing data shows that activity of 1 depends on activation of

HIF-1a, which provides a possible explanation why activity of

was restricted to cells in

HIF-1a positive areas. Further support of this hypothesis is the lack of increase of HIF-1a

positive cells in the tumors treated according to

Schedule B

and

. Since

did not affect

HIF-1a levels in U251 cells in tissue culture, it is likely that numbers of HIF-1a positive

cells were reduced as they went through apoptosis. Finally, increased reliance of cancer cells

on proximity to vasculature once again suggests their hindered ability to adapt to low partial

oxygen pressures, which is the main function of the hypoxic response. It is worth noting

that, despite the fact that more blood vessels exist in proximity of necrosis, the perinecrotic

areas are not representative of the whole tumor. These areas contain only a small fraction of

tumor area and inherently contain few blood vessels, which leads to necrosis. The transient

induction of HIF-1a levels in briefly-treated GBM39 tumors indicates a likely increase in

tumor hypoxia, or a change in HIF-1a protein levels by direct action of

. The latter

possibility is unlikely as

had no effect on HIF-1a levels in tissue culture. The apparent

disparity in HIF-1a levels between the GBM39 tumors treated according to

Schedule A

, and

other regimens, could be due to two factors: concentration of

was insufficient to induce

apoptosis of tumor cells in

Schedule A

, or overall level of hypoxia in these smaller tumors

(Supplementary Figure 8C) was too low to induce sensitivity to

. The latter hypothesis is

supported by comparable HIF-1a levels in non-necrotic regions of GBM39 tumors from a

group treated according to

Schedule C

(Supplementary Figure 7A) and by tendency of

smaller tumors to be less hypoxic (

). Reduced proliferation upon treatment with

is likely

a combination of both reduced MVD and a direct effect of 1 on cancer cells. The former

hypothesis is supported by the dependence of proliferation on distance from necrosis, while

the latter – by a reduction in Ki-67 in non-necrotic areas that are presumably sufficiently

vascularized for unrestricted cell division.

Effects on gene expression

Compound

reduced mRNA expression of a panel of genes involved in angiogenesis and

metastasis. Many of the affected genes carry important functions in tumor adaptation to

hypoxia – for example Platelet-derived growth factor subunit B (

PDGFB

) and

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Angiopoietin-1 (

ANGPT1

) are involved in blood vessel maturation (

), whereas Lysyl

Oxidase (

LOX

) or

RHOC

were involved in metastasis (

). Other factors significantly

affected by

, included:

FLT1, NLGN1, HOXB9, NEDD9, MMP2

and

MMP14

, and a

mouse Angiopoietin receptor,

Tek

. We also observed a slight elevation (1.4-fold) in both

mRNA expression and serum concentration of VEGFA, despite a visible downregulation in

tissue culture (

). Reduction in microvessel density typically leads to decreased oxygen

pressure and increased expression of proangiogenic factors, including VEGFA (

Interestingly, recent studies show that regulation of VEGFA activity can be HIF-1-

independent (

). However, it is also possible that VEGFA expression would be higher

had it not been for treatment with

. Importantly, elevated expression of VEGFA did not

prevent

from exerting antiangiogenic effect, suggesting its mechanism of action could be

VEGF-independent.

Conclusions

In summary, this study demonstrates that Py-Im polyamide

interferes with the tested

endpoints of hypoxic response in tumors: it inhibits prometastatic and proangiogenic gene

expression, induces tumor cell apoptosis selectively in HIF-1a positive areas and decreases

nuclear density, proliferative index and microvessel density of tumors. The RNA-sequencing

data revealed that

regulates genes differentially in cells with induced and basal levels of

HIF-1a, providing a potential explanation for selective induction of apoptosis in HIF-1a

positive cells. This compound could be useful in treatment of hypoxia-related disease as it

distributes to tissues, has favorable pharmacokinetics, and antitumor and antiangiogenic

activity in two different xenografts.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

The authors thank Caltech OLAR for technical assistance with animal experiments and TPCL (UCLA) for assisting

with IHC. The authors thank Dr. Nora Rozengurt (UCLA) for helpful suggestions, discussion and preliminary

pathological evaluation of tumor sections. We thank Drs. Nicholas Nickols and Bogdan Olenyuk for helpful

discussions and suggestions.

Financial support

: This work was supported by the NIH Grant GM-51747. J.O.Szablowski. was supported by NIH

GM-51747. J.A.Raskatov received postdoctoral support from the Alexander von Humboldt foundation.

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Figure 1. Chemical structure and biological activity of Py-Im polyamides binding HRE sequence

A) Chemical structures and ball-and-stick representation of the Py-Im polyamides

–

. N-

methyl-pyrrole, N-methyl-imidazole and chlorothiophene are represented as open circles,

filled circles and squares, respectively. B) Our previous studies (

) indicated that compound

can bind to HRE-containing sequence with sub-nanomolar (k

> 10

) affinity and displace

HIF-1 complex from VEGFA and CA9 promoters in U251 cells in Chromatin Immuno-

Precipitation assay (ChIP). C) Gene expression regulation by

(1 μM) in U251 cells with

elevated levels of HIF-1a (treatment with 300 μM Deferoxamine (DFO)) or native levels of

HIF-1a (PBS treatment). Cells were treated either with PBS or

for 48 hours, and then with

either PBS or DFO for 16 hours. Gene expression changes for each sample were obtained by

normalizing a polyamide-treated sample with the appropriate non-treated control – cells

treated with PBS and

were normalized to cells treated with PBS only, and cells treated

with both

and DFO were normalized to cells treated with DFO. Overall,

regulates

expression of a distinct set of genes when cells are co-treated with DFO and

. D)

Hierarchical clustering (Euclidian distance, complete linkage) of genes changed at least

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three-fold as compared to the state untreated with

. Gene expression changes expressed as

Log

values. Three biological replicates were analyzed.

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Figure 2. Polyamide 1 inhibits tumor growth and vascularization after systemic administration

A) Serum concentration of

after subcutaneous injection. The C57BL6 mice were injected

with

subcutaneously into interscapular area, after which we drew blood via retroorbital

collection (n=4 per time point). B) Nuclear uptake of

in U251 cells, counterstained with a

DNA-binding Hoechst 33342 fluorophore. C) Final masses and growth curve of s.c. U251

xenografts derived from animals subjected to

Schedule A

(n=7,8, for vehicle and treated

groups; for volumes, measurements at the 3rd an 5th day contain 5 data points.) D) Final

masses and growth curve of U251 xenografts (

Schedule B

, n=10 per condition). E) Mice

harboring xenografts were treated with

according to

Schedule A

and their MVD score was

measured using anti-mouse CD31 immunostaining of tumor sections (For U251 n=7,8 for

vehicle, and treated groups). F) Prolonged treatments, according to

Schedule B

in U251

xenografts led to comparable decrease in MVD. G) Apoptosis in blood vessels was

determined by double-staining of Cleaved Caspase-3 (CC3) and mouse CD31 of GBM39

tumor sections treated according to

Schedule C

. Both vehicle and polyamide-

treated

samples exhibited low levels of blood vessel apoptosis and the differences between the

groups were not significant (p=0.37, n=5 per condition). H) An in vitro angiogenesis assay -

matrigel tube formation assay using HUVECs - shows treatment with

(5 μM) over 48h has

no effect on endothelial tube formation. Arrows denote injections. Error bars are 95% CI for

growth curves and minimum-maximum for other graphs.

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