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. 2022 Aug 31:13:906357.
doi: 10.3389/fimmu.2022.906357. eCollection 2022.

Lipopolysaccharide sensitizes the therapeutic response of breast cancer to IAP antagonist

Xin Liu  1   2 , Jimmy J Yao  1   3 , Zhongxuan Chen  1   3 , Wei Lei  1   4 , Rong Duan  1 , Zhenqiang Yao  1
Affiliations

Lipopolysaccharide sensitizes the therapeutic response of breast cancer to IAP antagonist

Xin Liu et al. Front Immunol. .

Abstract

Inhibitor of apoptosis protein (IAP) is a class of E3 ubiquitin ligases functioning to support cancer survival and growth. Many small-molecule IAP antagonists have been developed, aiming to degrade IAP proteins to kill cancer. We have evaluated the effect of lipopolysaccharide (LPS), a component of the bacterial outer membrane, on IAP antagonists in treating breast cancer in a mouse model to guide future clinical trials. We show that LPS promotes IAP antagonist-induced regression of triple-negative breast cancer (TNBC) from MDA-MB-231 cells in immunodeficient mice. IAP antagonists such as SM-164, AT-406, and BV6, do not kill MDA-MB-231 cells alone, but allow LPS to induce cancer cell apoptosis rapidly. The apoptosis caused by LPS plus SM-164 is blocked by toll-like receptor 4 (TLR4) or MyD88 inhibitor, which inhibits LPS-induced TNFα production by the cancer cells. Consistent with this, MDA-MB-231 cell apoptosis induced by LPS plus SM-164 is also blocked by the TNF inhibitor. LPS alone does not kill MDA-MB-231 cells because it markedly increases the protein level of cIAP1/2, which is directly associated with and stabilized by MyD88, an adaptor protein of TLR4. ER+ MCF7 breast cancer cells expressing low levels of cIAP1/2 undergo apoptosis in response to SM-164 combined with TNFα but not with LPS. Furthermore, TNFα but not LPS alone inhibits MCF7 cell growth in vitro. Consistent with these, LPS combined with SM-164, but not either of them alone, causes regression of ER+ breast cancer from MCF7 cells in immunodeficient mice. In summary, LPS sensitizes the therapeutic response of both triple-negative and ER+ breast cancer to IAP antagonist therapy by inducing rapid apoptosis of the cancer cells through TLR4- and MyD88-mediated production of TNFα. We conclude that antibiotics that can reduce microbiota-derived LPS should not be used together with an IAP antagonist for cancer therapy.

Keywords: MyD88; TNF-α; Tolllike receptor 4; apoptosis; breast cancer; inhibitor of apoptosis protein; lipopolysaccharide.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
LPS triggers IAP antagonist-induced apoptosis of MDA-MB-231 cells. 1 × 105 MDA-MB-231 cells were cultured in 60-mm dishes for 24 h followed by treatment overnight with indicated dose of SM-164, AT-406, or BV6 −/+ LPS. AnnV+PI−/+ apoptotic cells were analyzed by flow cytometry. The data were from three repeat samples in one experiment. *p < 0.05 and **p < 0.01 between the indicated groups. The experiments were repeated three times with similar results. LPS, lipopolysaccharide; IAP, inhibitor of apoptosis protein.
Figure 2
Figure 2
LPS, given together with an IAP antagonist, induces the regression of TNBC in mouse model. (A) 2 × 104 MDA-MB-231 cells were injected into the second left mammary fat pad of female NSG mice. From the third day, the mice were treated with vehicle, 1 μg of LPS, 3 mg/kg of SM-164 (SM), or their combination for 4 weeks. The mice were euthanized, and the tumors were collected to scale their weight. (B) 2 × 105 MDA-MB- 231 cells were injected into female NSG mice as in panel (A). After 2 weeks, when the tumors were visible, the mice were treated with vehicle, 3 mg/kg of SM, or 10 mg/ml of BV6 alone or in combination with 1 μg of LPS for 3 weeks. The tumor weight was determined as in panel (A). Six to seven in each group. *p < 0.05, **p < 0.01. NS, Not significant. LPS, lipopolysaccharide; IAP, inhibitor of apoptosis protein; TNBC, triple-negative breast cancer.
Figure 3
Figure 3
LPS triggers SM-164-induced apoptosis of MDA-MB-231 cells through TLR4. MDA-MB-231 cells were treated overnight with 100 ng/ml of LPS and SM-164 (SM) 3 nM, plus indicated dose of TLR4 inhibitor (TLR4i). AnnV+PI−/+ apoptotic cells were analyzed by flow cytometry. The data were from three repeat samples in one experiment. **p < 0.01 between the indicated groups. The experiments were repeated three times with similar results. LPS, lipopolysaccharide.
Figure 4
Figure 4
Inhibition of MyD88 blocks LPS triggering SM-164-induced MDA-MB-231 cell apoptosis. 1 × 105 MDA-MB-231 cells were cultured in 60-mm dishes for 24 h. The cells were then treated with vehicle, SM-164 (3 nM) + LPS (100 ng/ml), or SM-164+LPS+ indicated dose of MyD88 inhibitor 4210. AnnV+PI−/+ apoptotic cells were analyzed by flow cytometry. The data were from three repeat samples in one experiment. *p < 0.05 and **p < 0.01 between the indicated groups. The experiments were repeated three times with similar results. LPS, lipopolysaccharide.
Figure 5
Figure 5
MyD88 associates with and stabilizes cIAP proteins. MDA-MB-231 cells at sub-confluence were treated with LPS (100 ng/ml) or its combination with SM-164 (3 nM) −/+ TLR4i (5 μM) (A) and were treated with LPS (100 ng/ml) −/+ MyD88 inhibitor 4210 (10 μM) (B) for 8 h; 20 μg of whole-cell lysate protein from each sample was used to test protein levels of MyD88, cIAP1/2, and β-actin by Western blotting (WB). The quantified WB data were from five (A) or three (B) repeats. *p < 0.05 and **p < 0.01 between the indicated groups. (C) Cell lysate protein measuring 300 μg from samples, as in panel B, was immunoprecipitated with anti-MyD88 antibody −/+ 10 μM of MyD88 inhibitor 4210. In addition, one sample was incubated with normal IgG. The pulldown proteins were used to test cIAP1/2 and MyD88 by WB. The experiments were repeated three times with similar results.
Figure 6
Figure 6
Autocrine TNFα mediated by TLR4 is critical for LPS triggering SM-164-induced apoptosis of MDA-MB-231 cells. (A) MDA-MB-231 cells were treated overnight with SM-164 (SM) 3 nM plus either 100 ng/ml of LPS or 1 ng/ml TNFα or with their combination with TNFR: Fc. AnnV+PI−/+ apoptotic cells were analyzed by flow cytometry. The data were from three repeat samples in one experiment. *p < 0.05 and **p < 0.01 between the indicated groups. (B) The culture medium of MDA-MB-231 cells, as in panel A and Figures 3 and 4 , were collected. TNFα levels were tested by ELISA. The data were from three repeats. *p < 0.05 and **p < 0.01 between the indicated groups. LPS, lipopolysaccharide.
Figure 7
Figure 7
LPS in combination with an IAP antagonist induces ER+ breast cancer regression in mouse model. (A) 2 × 105 MCF7 cells were cultured in 60-mm dishes for 24 h followed by treatment overnight with vehicle, 100 ng/ml of LPS, or 1 ng/ml of TNFα alone or each of them in combination with 3 nM of SM-164. AnnV+PI−/+ apoptotic cells were analyzed by flow cytometry. The data were from three repeat samples in one experiment. *p < 0.05 and **p < 0.01 between the indicated groups. (B) MDA-MB-231 or MCF7 cells at sub-confluent in 60-mm dishes were treated with vehicle, LPS, and/or TNFα for 8 h; 20 μg of cell lysate protein from each sample was used to test protein levels of cIAP1/2 by WB. (C) 4 × 104 MCF7 cells in a well of 6-well plates were treated with vehicle, LPS, or TNFα for 8 days. The cells in each well were digested with 0.25% of trypsin to count cell number. Three repeats for each treatment. **p < 0.01 vs. Veh or LPS. (D) 2 × 105 MCF7 cells were injected into the second left mammary fat pad of a female NSG mouse. On days 3 and 15 after cancer injection, the mice were treated with vehicle, 1 μg of LPS, 3 mg/kg of SM-164 (SM), or their combination for 4 weeks. The mice were euthanized, and the tumors were collected to scale their weight, eight in each group *p < 0.05, **p < 0.01. LPS, lipopolysaccharide; IAP, inhibitor of apoptosis protein; WB, Western blotting.
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