The Human Intermediate Prolactin Receptor I-tail Contributes Breast Oncogenesis by Targeting Ras/MAPK Pathway.

Prolactin and its receptor (PRLr) in humans are significantly involved in breast cancer pathogenesis. The intermediate form of human PRLr (hPRLrI) is produced by alternative splicing and has a novel 13 amino acid tail ("I-tail") gain. hPRLrI induces significant proliferation and anchorage-independent growth of normal mammary epithelia in vitro when coexpressed with the long form hPRLr (hPRLrL). hPRLrL and hPRLrI coexpression is necessary to induce the transformation of mammary epithelia in vivo. The I-tail is associated with the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Treatment with the neural precursor cell expressed developmentally downregulated protein 8-activating enzyme inhibitor pevonedistat resulted in increased hPRLrL and the death of breast cancer cells. The goal of this study was to determine the function of the hPRLrI I-tail in hPRLrL/hPRLrI-mediated mammary transformation. hPRLrL/hPRLrI and hPRLrL/hPRLrIΔ13 (I-tail removal mutant) were delivered to MCF10AT cells. Cell proliferation was decreased when hPRLrI I-tail was removed. I-tail deletion decreased anchorage-independent growth and attenuated cell migration. The I-tail was involved in Ras/MAPK signaling but not PI3K/Akt signaling pathway as shown by western blot. I-tail removal resulted in decreased hPRLrI stability. RNA-sequencing data revealed that I-tail removal resulted in differential gene expression induced by prolactin. Ingenuity Pathway Analysis revealed that the activity of ERK was attenuated. Treatment of breast cancer cells with ERK1/2 inhibitor ulixertinib resulted in decreased colony-forming ability and less proliferation. These studies suggest that the hPRLrI I-tail contributed to breast oncogenesis and may be a promising target for the development of new breast cancer therapies.

Pharmacologic Inhibition of SHP2 Blocks Both PI3K and MEK Signaling in Low-epiregulin HNSCC via GAB1.

Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.

Exploitation of Sulfated Glycosaminoglycan Status for Precision Medicine of Triplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Unmasking BCL-2 Addiction in Synovial Sarcoma by Overcoming Low NOXA.

Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.

Catastrophic ATP loss underlies a metabolic combination therapy tailored for MYCN-amplified neuroblastoma.

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an "Achilles' heel" and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors.

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

MYCN-Amplified Neuroblastoma Is Addicted to Iron and Vulnerable to Inhibition of the System Xc-/Glutathione Axis.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. SIGNIFICANCE: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.

Syndecan-1 facilitates breast cancer metastasis to the brain.

PURPOSE: Although survival rates for patients with localized breast cancer have increased, patients with metastatic breast cancer still have poor prognosis. Understanding key factors involved in promoting breast cancer metastasis is imperative for better treatments. In this study, we investigated the role of syndecan-1 (Sdc1) in breast cancer metastasis. METHODS: To assess the role of Sdc1 in breast cancer metastasis, we silenced Sdc1 expression in the triple-negative breast cancer human MDA-MB-231 cell line and overexpressed it in the mouse mammary carcinoma 4T1 cell line. Intracardiac injections were performed in an experimental mouse metastasis model using both cell lines. In vitro transwell blood-brain barrier (BBB) and brain section adhesion assays were utilized to specifically investigate how Sdc1 facilitates brain metastasis. A cytokine array was performed to evaluate differences in the breast cancer cell secretome when Sdc1 is silenced. RESULTS: Silencing expression of Sdc1 in breast cancer cells significantly reduced metastasis to the brain. Conversely, overexpression of Sdc1 increased metastasis to the brain. We found that silencing of Sdc1 expression had no effect on attachment of breast cancer cells to brain endothelial cells or astrocytes, but migration across the BBB was reduced as well as adhesion to the perivascular regions of the brain. Loss of Sdc1 also led to changes in breast cancer cell-secreted cytokines/chemokines, which may influence the BBB. CONCLUSIONS: Taken together, our study demonstrates a role for Sdc1 in promoting breast cancer metastasis to the brain. These findings suggest that Sdc1 supports breast cancer cell migration across the BBB through regulation of cytokines, which may modulate the BBB. Further elucidating this mechanism will allow for the development of therapeutic strategies to combat brain metastasis.

The Laminin-α1 Chain-Derived Peptide, AG73, Binds to Syndecans on MDA-231 Breast Cancer Cells and Alters Filopodium Formation.

Breast cancer is one of the most common forms of cancer affecting women in the United States, second only to skin cancers. Although treatments have been developed to combat primary breast cancer, metastasis remains a leading cause of death. An early step of metastasis is cancer cell invasion through the basement membrane. However, this process is not yet well understood. AG73, a synthetic laminin-α1 chain peptide, plays an important role in cell adhesion and has previously been linked to migration, invasion, and metastasis. Thus, we aimed to identify the binding partner of AG73 on breast cancer cells that could mediate cancer progression. We performed adhesion assays using MCF10A, T47D, SUM1315, and MDA-231 breast cell lines and found that AG73 binds to syndecans (Sdcs) 1, 2, and 4. This interaction was inhibited when we silenced Sdcs 1 and/or 4 in MDA-231 cells, indicating the importance of these receptors in this relationship. Through actin staining, we found that silencing of Sdc 1, 2, and 4 expression in MDA-231 cells exhibits a decrease in the length and number of filopodia bound to AG73. Expression of mouse Sdcs 1, 2, and 4 in MDA-231 cells provides rescue in filopodia, and overexpression of Sdcs 1 and 2 leads to increased filopodium length and number. Our findings demonstrate an intrinsic interaction between AG73 in the tumor environment and the Sdcs on breast cancer cells in supporting tumor cell adhesion and invasion through filopodia, an important step in cancer metastasis.

NSG Mice Provide a Better Spontaneous Model of Breast Cancer Metastasis than Athymic (Nude) Mice.

Metastasis is the most common cause of mortality in breast cancer patients worldwide. To identify improved mouse models for breast cancer growth and spontaneous metastasis, we examined growth and metastasis of both estrogen receptor positive (T47D) and negative (MDA-MB-231, SUM1315, and CN34BrM) human breast cancer cells in nude and NSG mice. Both primary tumor growth and spontaneous metastases were increased in NSG mice compared to nude mice. In addition, a pattern of metastasis similar to that observed in human breast cancer patients (metastases to the lungs, liver, bones, brain, and lymph nodes) was found in NSG mice. Furthermore, there was an increase in the metastatic burden in NSG compared to nude mice that were injected with MDA-MB-231 breast cancer cells in an intracardiac experimental metastasis model. This data demonstrates that NSG mice provide a better model for studying human breast cancer metastasis compared to the current nude mouse model.