Molecular analysis of the selective pro-apoptotic effect of BN107 on estrogen receptor negative breast cancer cells

Ruth Chu, Xiaoyue Zhao, Chandi Griffin, Mark Shoemaker, Mary Tagliaferi, Dale Leitman, Isaac Cohen, Emma Shtivelman, and Sylvia FongBionovo Inc., Emeryville, CA

Abstract # 551708

Background

• Mitochondrial-mediated apoptosis appears to be the major cellular pathway mediating the growth inhibitory effect of BN107.

• Cholesterol depletion and disruption of lipid raft-mediated signaling might be responsible for the pro-apoptotic effect of BN107.

• Estrogen receptor status correlates with BN107 sensitivity. Specifically, ER- breast cancer lines that have mesenchymal characateristics, are highly sensitive to BN107. Conversely, when ER expression is exogenously introduced or induced, the cells exhibit far less sensitivity to BN107.

• Studies are underway to investigate the protective role of ER in lipid-raft disruption induced by BN107; as well as to identify active components in the extract responsible for its anti-tumor effect.

1. Rugo et al. (2006) Breast Cancer Res Treat, 105(1):17-28. 2. Fong et al. (2008) Cancer Biol Ther,7(4): 577-586.3. Campbell et al. (2002) Anticancer research, 22, 3843-3853.

0

20

40

60

80

Caspase 3 Caspase 9% cells with activated caspase

ControlBN107

Annexin V PI staining

ER Her2

SKBr3 ++ - +

Hs578T ++++ - +

MDA-MB-468 ++ - -

MDA-MB-231 ++++ - -

MDA-MB-453 ++++ - +

MCF10A +++ - -

IMR90 ++++ - -

MDA-MB-361 -/+ + +

BT474 -/+ + +

MCF7 - + -

References

Results ResultsResults Results

Summary

BN107

0 4 8 16 BN107 Rx (hr)

Cytochrome C

GAPDH

Results

Hs578T (ER-) MCF7 (ER+)

- Apoptosis

- Cell cycle

- Cholesterol synthesis

- Oxidative response

- MAPK signaling

- Acute stress response

- Ah receptor signaling

- IGF1 receptor signaling

- MAPK signaling

- Cell growth

- Acute stress response

MDA231 Cont

MDA231 BN107

MDA231 BN107+CHL

MCF7 ContMCF7 BN107

0

20

40

60

80

100

120

% Survival

% Live cells

Control

BN107

BN107+CHL

BN107+LDL

BN107+GOH

BN107+FOH

Taxol

Taxol+CHL

MCF7 MDA-MB231

p-mTOR (Ser2448)mTORRICTORRAPTORp-S6K (Thr389)

Total 4EBP

GAPDH

- - + + BN107, 4hr

- + - + LDL (50 g/ml)pS6K (Thr389)Total S6K

p4EBP (Ser65)

Total 4EBP

020406080

100120

LacZ ERaLive Cells, % Control

Control

BN107

0306090

LacZ ERa

WISP2

Rela

tive

expr

essio

n

050

100150200250300350400450

LR Non LR plasmamembrane

Total

g of CHL in fractions

MDA231 Cont

MDA231 BN107

MCF7 Cont

MCF7 BN107

LR Non-LR PMGM-1 TR

Annexin V

Annexin V

PICont BN107

CAV1

CD44 0

20

40

60

80

100

120

% Live cells, % Control

Control

DMSO

TsA

PIControl

MDA231 Hs578TBN107 - + - +

S6K

0 1 3 6 16 0 1 3 6 16 BN107 Rx (hr)

p-4EBP (Ser65)

Transferrin Receptor

ER

GAPDH

+ + BN107 + + E2 (10 nM)

GM-1

CD44

CAV1

0 1 3 6 16 0 1 3 6 16 BN107 Rx (hr)

- + + BN107 Rx

- - + CHL

LR

Non-LR PM

p-4EBP (Ser65)

4EBP

Flotillin 1

TR

• Despite advances in early detection and adjuvant therapy have made a favorable impact on survival, current regimens lead to toxic side effects and are mostly ineffective against metastatic breast cancer. Currently, patients with estrogen receptor (ER)-negative/progesterone receptor (PR)-negative/HER-2-negative tumors still present a therapeutic challenge for the oncologists. Therefore, novel and effective therapies with minimal toxicities are urgently needed for this patient population.

• Botanical medicines are frequently sought and used by cancer patients, yet few studies have evaluated their safety and efficacy as well as their mechanisms of action.

• Bionovo Inc. has a pipeline of anti-breast cancer products in development. We have recently completed a phase 1/1B clinical trial for BZL101, a drug candidate for metastatic breast cancer, in a heavily pretreated patient population with metastatic breast cancer and showed that it had a favorable toxicity profile and demonstrated encouraging clinical efficacy (Rugo et al. 2006). We are currently enrolling patients for phase 2 clinical testing. Analysis of mechanism of action of BZL101 demonstrated the selectivity of BZL101 in targeting breast cancer cells while sparing non-transformed cells (Fong, et al. 2008).

• Here, we present results of analysis on the mechanism of action of BN107, another anti-breast cancer product in development, ,for its pro-apoptotic effect on breast cancer cells. BN107 is an aqueous extract of the fruit of Gleditsia sinensis Lam. It has been shown to exhibit strong growth inhibition across various breast cancer cell lines (Campbell et al. 2002).

– BN107 appears to exert its growth inhibition properties on breast cancer cells via the mitochondrial apoptotic machinery.

– BN107 induces rapid alterations in mTOR signaling pathway and in cholesterol (CHL) metabolism. Co-administering CHL and BN107 abolishes its pro-apoptotic effect, as well as restores the mTOR signaling pathway.

– The disruption of CHL-rich lipid raft might be mediating the pro-apoptotic effect of BN107.

– Absence of estrogen receptor (ER) in the cells correlates with sensitivity to BN107. Introduction of ER expression into a breast cancer line results in protection from the pro-apoptotic effect of BN107. Consistently, treating ER- breast cancer cells with a differentiating agent (trichostatin A), also confers resistance to BN107. Transcriptomic analysis comparing sensitive (ER-) and insensitive (ER+) lines treated with BN107 reveals distinct patterns of gene expression that might be responsible for the differential sensitivity.

Results

MCF7 MDA-MB-231

BN107 induces apoptosis in breast cancer cellsA. B.

C. D.

Figure 1. BN107 induces apoptosis via mitochondrial machinery in breast cancer cells, assessed by A. Annexin V-PI staining, B. DNA fragmentation, C. Western blot showing Cytochrome C released in cytosol, and, D. Activation of caspases 3 and 9. Treatment with BN107 also leads to dissipation of mitochondrial transmembrane potential (data not shown). Hs578T cells were treated with BN107 (70 g/ml) and harvested after 18 (A), 8 (B), or 3 (D) hours of treatment.

Table 1. Cells without ER are more sensitive to BN107 induced apoptosis, while Her2 status appears not correlative with BN107 sensitivity. Cells were treated with BN107 and harvested after 18 hours for analysis of Annexin V/PI binding. The summary shown is a result of 3 independent experiments.

BN107 induces expression of distinct sets of genes and alters mTOR signaling in sensitive vs insensitive breast cancer cells

Table 2. Cellular/signaling pathways induced by BN107 treatment, based on Ingenuity Pathway Analysis (IPA) of microarray data generated using BN107 sensitive (Hs578T) and insensitive (MCF7) cells treated with BN107 for 4 hr.

Figure 2. Analysis of proteins involved in mTOR signaling pathway. MDA231 (sensitive) and MCF7 (insensitive) cells were treated with BN107 (70 g/ml) and harvested at indicated time points. Exogenous cholesterol protects cells from BN107 induced apoptosis and restores mTOR signaling pathway

A. B.

Figure 3. Cholesterol specifically protects cells from BN107 induced death and restores mTOR signaling. A. MDA-MB231 cells were treated with 70 g/ml BN107 alone or with 500 M cholesetrol (CHL), 50 g/ml LDL, 50 M of isoprenoid precursors (geranylgeraniol, GOH or farnesol, FOH) for 18 hours. The chart shows percentage of live cells (annexin V-, PI-). B. Western blot analysis of mTOR downstream substrates in cells treated with BN107 and +/- LDL.

BN107 depletes CHL and disrupts lipid raft (LR) and LR-mediated mTOR signalingA.

B.

C. D.

Figure 4 . In ER – cell line, the cholesterol content and the localization of lipid raft (LR) resident proteins, as well as LR-mediated mTOR signaling are disrupted by BN107 treatment. A. CHL content in LR, Non-LR plasma membrane (PM) fractions and total lysate. B.. Immunofluorescence staining of caveolin 1 (CAV1) and CD44 (green) in cells treated with 70 g/ml of BN107 for 4 hrs and fixed in 4% paraformaldehyde. C. Dot plot analysis of GM-1 (marker for LR fractions) and transferrin receptor (TR, marker for non-LR plasma membrane fractions). D. Western analysis of total lysate (upper panel) and LR or non-LR PM fractions (lower panel). MDA-MB231 or MCF7 cells were treated with 60 g/ml BN107 (± 500 M CHL) for 3 hr. Various fractions were collected after separation of triton-X100 soluble and insoluble proteins with density gradient centrifugation (A, C, D). Fractions were spotted directly from fractions (C), or were precipitated to load the same amount of protein (D).

Expression of ER in ER- breast cancer cell line confers resistance to BN107A. B.

C.

Figure 5. ER expression rescues MDA-MB231 cells from BN107 induced apoptosis. A. MDA-MB231 cells infected with LacZ or ER virus were treated with BN107 for 18 hrs in the presence of estrogen (E2, 10 nM) and analyzed with Annexin/PI binding. The chart shows percentage of Annexin- PI- (live) cells. B. Real time qRTPCR analysis of WISP2 expression, a downstream target of ER, indicating functional ER status. C. MDA-MB-231 cells were treated with a differentiating histone deacetylase inhibitor, tricostatin A (TsA, 50nM ) or DMSO for 2 days. The cells were then treated with BN107 and analyzed with Annexin/PI binding as in A.