Deborah H. Anderson

Lung squamous cell carcinoma (LUSC) is a difficult cancer to treat, with few targeted therapies to improve its poor prognosis. The goal of this study was to use a drug repurposing strategy to evaluate and compare drug sensitivities using 2D adherent and 3D spheroid models of NCI-H2170 LUSC cells. Both 2D adherent and 3D spheroid models were used to grow NCI-H2170 lung squamous cell carcinoma cells and evaluate their sensitivity to a large library of food and drug administration (FDA)-approved drugs, including many not typically used as anti-cancer agents. Cell death was assessed in the 2D adherent models, and for the top drugs half maximal effective concentration (EC ₅₀ ) values were determined. For the 3D spheroid models, drugs reducing spheroid size after 4 days of treatment were identified. There were 263 drugs that reduced the cell viability to <20% when cells were grown in 2D in 10 µM drug. When grown in 3D the cells were generally more drug resistant, with 87 drugs capable of reducing spheroid volume when grown over 4 days in 10 µM drug. Interestingly, 60 drugs proved effective in both model systems including many drugs that typically associated with anti-cancer properties. Of these 60, four were further found to have selective effects towards metastatic NCI-H2170 cells as compared to a much less metastatic matched cell line expressing the metastasis suppressor CREB3L1, in both 2D and 3D model systems. These included the hypoxia-inducible factor 1-alpha (HIF-1α inhibitor 2-methoxyestradiol, and three anti-infection agents (cetylpyridinium chloride, chlorhexidine-2HCl, zinc pyrithione). Conclusions: These results suggest that the HIF-1α inhibitor 2-methoxyestradiol, and three anti-infection agents (cetylpyridinium chloride, chlorhexidine-2HCl, zinc pyrithione) may be effective for metastatic LUSCs.

Women with metastatic breast cancer have a disheartening 5-year survival rate of only 28%. CREB3L1 (cAMP responsive element binding protein 3 like 1) is a

The lack of targeted therapies for triple-negative breast cancer (TNBC) contributes to their high mortality rates and high risk of relapse compared to other subtypes of breast cancer. Most TNBCs (75%) have downregulated the expression of CREB3L1 (cAMP-responsive element binding protein 3 like 1), a transcription factor and metastasis suppressor that represses genes that promote cancer progression and metastasis. In this report, we screened an FDA-approved drug library and identified four drugs that were highly cytotoxic towards HCC1806 CREB3L1-deficient TNBC cells. These four drugs were: (1) palbociclib isethionate, a CDK4/6 inhibitor, (2) lanatocide C (also named isolanid), a Na+/K+-ATPase inhibitor, (3) cladribine, a nucleoside analog, and (4) homoharringtonine (also named omacetaxine mepesuccinate), a protein translation inhibitor. Homoharringtonine consistently showed the most cytotoxicity towards an additional six TNBC cell lines (BT549, HCC1395, HCC38, Hs578T, MDA-MB-157, MDA-MB-436), and several luminal A breast cancer cell lines (HCC1428, MCF7, T47D, ZR-75-1). All four drugs were then separately evaluated for possible synergy with the chemotherapy agents, doxorubicin (an anthracycline) and paclitaxel (a microtubule stabilizing agent). A strong synergy was observed using the combination of homoharringtonine and paclitaxel, with high cytotoxicity towards TNBC cells at lower concentrations than when each was used separately.

Women with metastatic breast cancer have a disheartening 5-year survival rate of only 28%. CREB3L1 (cAMP-responsive element binding protein 3 like 1) is a metastasis suppressor that functions as a transcription factor, and in an estrogen-dependent model of rat breast cancer, it repressed the expression of genes that promote breast cancer progression and metastasis. In this report, we set out to determine the expression level of CREB3L1 across different human breast cancer subtypes and determine whether CREB3L1 functions as a metastasis suppressor, particularly in triple negative breast cancers (TNBCs). CREB3L1 expression was generally increased in luminal A, luminal B and HER2 breast cancers, but significantly reduced in a high proportion (75%) of TNBCs. Two luminal A (HCC1428, T47D) and two basal TNBC (HCC1806, HCC70) CREB3L1-deficient breast cancer cell lines were characterized as compared to their corresponding HA-CREB3L1-expressing counterparts. HA-CREB3L1 expression significantly reduced both cell migration and anchorage-independent growth in soft agar but had no impact on cell proliferation rates as compared to the CREB3L1-deficient parental cell lines. Restoration of CREB3L1 expression in HCC1806 cells was also sufficient to reduce mammary fat pad tumor formation and lung metastases in mouse xenograft models of breast cancer as compared to the parental HCC1806 cells. These results strongly support a metastasis suppressor role for CREB3L1 in human luminal A and TNBCs. Further, the ability to identify the subset of luminal A (7%) and TNBCs (75%) that are CREB3L1-deficient provides opportunities to stratify patients that would benefit from additional treatments to treat their more metastatic disease.

BRK is, a non-receptor tyrosine kinase, overexpressed in approximately 85% of human invasive ductal breast tumors. It is not clear whether BRK expression correlates with breast cancer subtypes, or the expression has prognostic or diagnostic significance. Herein, we investigated the correlation of BRK with any breast cancer subtypes and clinicopathological significance of BRK expression in breast cancer. In this study, we examined BRK expression in 120 breast tumor samples and 29 breast cancer cell lines to explore the positive correlation between BRK and the expression of ERα. We used immunohistochemistry, RT-PCR, and immunoblotting to analyse our experimental samples. We demonstrate that estrogen induces BRK gene and protein expression in ER+ breast cancer cells. Over-expression of ERα in the ER-negative breast cancer cell line increased BRK expression, and knock-down of ESR1 in MCF7 cells reduced BRK levels. Further, we provide evidence that BRK is regulated by ERα signaling and the presence of ER antagonists (tamoxifen and fulvestrant) reduce the expression of BRK in ER-positive breast cancer cells. Finally, we demonstrate that the overall survival of ER-positive breast cancer patients is poor when their cancers express high levels of BRK. Our data indicate that BRK is a prognostic marker for ER+ breast cancers and provide a strong rationale for targeting BRK to improve patients’ survival.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in the regulation of cell signaling, proliferation, survival, migration and vesicle trafficking in normal cells and is frequently deregulated in many cancers. The p85α protein is the most characterized regulatory subunit of the class IA PI3Ks, best known for its regulation of the p110-PI3K catalytic subunit. In this review, we will discuss the impact of p85α mutations or alterations in expression levels on the proteins p85α is known to bind and regulate. We will focus on alterations within the N-terminal half of p85α that primarily regulate Rab5 and some members of the Rho-family of GTPases, as well as those that regulate PTEN (phosphatase and tensin homologue deleted on chromosome 10), the enzyme that directly counteracts PI3K signaling. We highlight recent data, mapping the interaction surfaces of the PTEN–p85α breakpoint cluster region homology (BH) domain, which sheds new light on key residues in both proteins. As a multifunctional protein that binds and regulates many different proteins, p85α mutations at different sites have different impacts in cancer and would necessarily require distinct treatment strategies to be effective.

Oncotarget | https://doi.org/10.18632/oncotarget.26432 Jeremy D.S. Marshall, Paul Mellor, Xuan Ruan, Dielle E. Whitecross, Stanley A. Moore, Deborah H. Anderson Insight into the PTEN – p85α interaction and lipid binding properties of the p85α BH domain

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its “switch regions” from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.