Jeff C. Liu

Identification of tumorsphere- and tumor-initiating cells in HER2/Neu-induced mammary tumors.

A variety of human malignancies, including breast cancer, are thought to be organized in a hierarchy, whereby a relatively minor population of tumor initiating cells (TIC) is responsible for tumor growth and the vast majority of remaining cells is nontumorigenic. Analysis of TICs in model systems of breast cancer would offer uniform and accessible source of tumor cells and the power of mouse genetics to dissect these rare cells. The HER2/Neu proto-oncogene is overexpressed in an aggressive form of human breast cancer. Mouse mammary tumor virus (MMTV)-Neu transgenic mice develop mammary tumors that mimic human HER2 subtype breast cancer. Here, we report on the functional identification of mouse HER2/Neu TICs that can induce tumors after transplantation into the mammary gland of recipient mice. Secondary tumors formed after injecting MMTV-Neu TICs resemble primary tumors in the original transgenic mice and are organized in a hierarchy containing TICs as well as their nontumorigenic descendants. To study MMTV-Neu TICs in vitro, we grew tumorspheres under nonadherent culture conditions. Tumorsphere forming units (TFU) capable of producing tumorspheres retained tumorigenic potential and were indistinguishable by several criteria from TICs. Interestingly, MMTV-Neu TICs and TFUs were committed to the luminal cell fate when induced to differentiate in vitro. Our data define reproducible characteristics of the MMTV-Neu TIC and TFU, which help to explain marker expression profiles of HER2-positive breast cancer. In addition, the similarity between TICs and TFUs in this system provides a rationale for TFU-based screens to target tumor-initiating cells in HER2(+) breast cancer.

Rb deletion in mouse mammary progenitors induces luminal-B or basal-like/EMT tumor subtypes depending on p53 status

Breast cancer is a highly heterogeneous disease, with several different subtypes being characterized by distinct histology, gene expression patterns, and genetic alterations. The tumor suppressor gene retinoblastoma 1 (RB1) is frequently lost in both luminal-B and triple-negative tumor (TNT; i.e., estrogen receptor-, progesterone receptor-, and human epidermal growth factor receptor 2-negative) breast cancer subtypes. However, a causal role for RB1 loss in different subtypes remains undefined. Here we report that deletion of Rb alone or together with its relative p107 in mouse mammary stem/bipotent progenitor cells induced focal acinar hyperplasia with squamous metaplasia. These lesions progressed into histologically diverse, transplantable mammary tumors with features of either luminal-B or TNT subtypes. The TNTs included basal-like tumors as well as tumors that exhibited epithelial-to-mesenchymal transition (EMT). The EMT-type tumors and a subset of the basal-like tumors, but not luminal-B-like tumors, expressed mutant forms of the tumor suppressor p53. Accordingly, targeted deletion of both Rb and p53 in stem/bipotent progenitors led to histologically uniform, aggressive, EMT-type tumors. Reintroduction of Rb into these tumor cells suppressed growth in vitro and tumor formation in vivo. These results establish a causal role for Rb loss in breast cancer in mice and demonstrate that cooperating oncogenic events, such as mutations in p53, dictate tumor subtype after Rb inactivation.

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.

Cooperation between Pik3ca and p53 mutations in mouse mammary tumor formation.

PIK3CA, which codes for the p110α catalytic subunit of phosphatidylinositol 3-kinase, is one of the most frequently mutated genes in human breast cancer. Here, we describe a mouse model for PIK3CA-induced breast cancer by using the ROSA26 (R26) knock-in system, in which targeted Pik3ca alleles can be activated through transgenic expression of Cre recombinase. We mated Pik3ca(H1047R) and Pik3ca(wt) knock-in lines with MMTV-Cre transgenics, which express Cre in mammary epithelium. Starting at approximately 5 months of age, female R26-Pik3ca(H1047R);MMTV-Cre mice, but not control R26-Pik3ca(wt);MMTV-Cre mice, developed mammary tumors, as well as lymphoid and skin malignancies. R26-Pik3ca(H1047R);MMTV-Cre mammary tumors were typically either adenosquamous carcinoma or adenomyoepithelioma. As p53 is the most commonly mutated gene in breast cancer, we tested for genetic interaction between Pik3ca(H1047R) and p53 loss-of-function mutations in R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mice. This led to decreased survival of double-mutant animals, which developed lymphoma and mammary tumors with rapid kinetics. Mammary tumors that formed in p53(loxP/+);MMTV-Cre conditional mutants were either poorly differentiated adenocarcinoma or spindle cell/EMT, whereas R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mammary tumors were mostly adenosquamous carcinoma or spindle cell/EMT indicating that double-mutant mice develop a distinct spectrum of mammary tumors. Thus, an oncogenic variant of PIK3CA implicated in multiple human breast cancer subtypes can induce a very diverse spectrum of mammary tumors in mice. Furthermore, Pik3ca(H1047R) shows cooperation with p53, which altered the specific tumors that formed. Thus, the two most frequently mutated genes in human breast cancer show cooperation in mammary tumor formation.

Combined deletion of Pten and p53 in mammary epithelium accelerates triple‐negative breast cancer with dependency on eEF2K

The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary‐specific deletion of Pten via WAP‐Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV‐Cre, which targets basal/luminal progenitors. Combined Pten‐p53 mutations accelerated formation of claudin‐low, triple‐negative‐like breast cancer (TNBC) that exhibited hyper‐activated AKT signaling and more mesenchymal features relative to Pten or p53 single‐mutant tumors. Twenty‐four genes that were significantly and differentially expressed between WAP‐Cre:Pten/p53 and MMTV‐Cre:Pten/p53 tumors predicted poor survival for claudin‐low patients. Kinome screens identified eukaryotic elongation factor‐2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53‐deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53‐deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin‐low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.

Seventeen-gene signature from enriched Her2/Neu mammary tumor-initiating cells predicts clinical outcome for human HER2+:ERα− breast cancer

Human Epidermal Growth Factor Receptor 2-positive (HER2+) breast cancer (BC) is a highly aggressive disease commonly treated with chemotherapy and anti-HER2 drugs, including trastuzumab. There is currently no way to predict which HER2+ BC patients will benefit from these treatments. Previous prognostic signatures for HER2+ BC were developed irrespective of the subtype or the hierarchical organization of cancer in which only a fraction of cells, tumor-initiating cells (TICs), can sustain tumor growth. Here, we used serial dilution and single-cell transplantation assays to identify MMTV-Her2/Neu mouse mammary TICs as CD24+:JAG1− at a frequency of 2–4.5%. A 17-gene Her2-TIC-enriched signature (HTICS), generated on the basis of differentially expressed genes in TIC versus non-TIC fractions and trained on one HER2+ BC cohort, predicted clinical outcome on multiple independent HER2+ cohorts. HTICS included up-regulated genes involved in S/G2/M transition and down-regulated genes involved in immune response. Its prognostic power was independent of other predictors, stratified lymph node+ HER2+ BC into low and high-risk subgroups, and was specific for HER2+:estrogen receptor alpha-negative (ERα−) patients (10-y overall survival of 83.6% for HTICS− and 24.0% for HTICS+ tumors; hazard ratio = 5.57; P = 0.002). Whereas HTICS was specific to HER2+:ERα− tumors, a previously reported stroma-derived signature was predictive for HER2+:ERα+ BC. Retrospective analyses revealed that patients with HTICS+ HER2+:ERα− tumors resisted chemotherapy but responded to chemotherapy plus trastuzumab. HTICS is, therefore, a powerful prognostic signature for HER2+:ERα− BC that can be used to identify high risk patients that would benefit from anti-HER2 therapy.

RB1 status in triple negative breast cancer cells dictates response to radiation treatment and selective therapeutic drugs.

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA+/CD24−/low/CD44+ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: interaction with IQ motif-containing factors.

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC50 for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA+/CD24-/low/CD44+ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.

RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

Preferential Killing of Breast Tumor Initiating Cells by N,N-Diethyl-2-[4-(Phenylmethyl)Phenoxy]Ethanamine/Tesmilifene

Purpose:N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) is thought to potentiate the antineoplastic effect of cytotoxic drugs. In a phase III randomized trial for metastatic breast cancer using doxorubicin with or without DPPE, addition of the latter resulted in a significant improvement in overall survival and a trend toward a difference in progression-free survival but, paradoxically, no difference in objective tumor response. Here we tested the hypothesis that DPPE targets breast tumor-initiating cells (TICs). Experimental Design: Human breast TICs from pleural effusions were identified as CD44+:CD24−/low cells by flow cytometry and functionally by their ability to form nonadherent spheres in culture. Mouse mammary TICs from two different models of breast cancer were identified as cells capable of initiating spheres in culture and secondary tumors following transplantation into the mammary gland of syngeneic mice. Results: We show that at physiologically attainable concentrations, treatment with DPPE alone reduced tumorsphere formation and viability of CD44+:CD24−/low breast cancer cells. The kinetics of killing varied for the different breast tumor cells and required continuous exposure to the drug. Whereas doxorubicin killed CD44+:CD24−/low and CD44−:CD24+ cells equally well, DPPE induced apoptosis preferentially in CD44+:CD24−/low cells. Treatment of Her2/Neu+ mammary tumor cells with DPPE in vitro efficiently killed TICs, as determined by flow cytometry and transplantation assays; DPPE further cooperated with doxorubicin to completely eradicate tumorigenic cells. Conclusions: Our results show that continuous treatment with DPPE alone directly targets breast TICs, and provide rationale to test for cooperation between DPPE and known drugs with efficacy toward breast cancer subtypes.