Search

Recurrent breast cancers often develop resistance to standard-of-care therapies. Identifying targetable factors contributing to cancer recurrence remains the rate-limiting step in improving long-term outcomes. In this study, we identify tumor cell-derived osteopontin as an autocrine and paracrine driver of tumor recurrence. Osteopontin promotes tumor cell proliferation, recruits macrophages, and synergizes with IL-4 to further polarize them into a pro-tumorigenic state. Macrophage depletion and osteopontin inhibition decrease recurrent tumor growth. Furthermore, targeting osteopontin in primary tumor-bearing female mice prevents metastasis, permits T cell infiltration and activation, and improves anti-PD-1 immunotherapy response. Clinically, osteopontin expression is higher in recurrent metastatic tumors versus female patient-matched primary breast tumors. Osteopontin positively correlates with macrophage infiltration, increases with higher tumor grade, and its elevated pathway activity is associated with poor prognosis and long-term recurrence. Our findings suggest clinical implications and an alternative therapeutic strategy based on osteopontin's multiaxial role in breast cancer progression and recurrence., (© 2024. The Author(s).)

Metabolic reprogramming, a hallmark of tumorigenesis, involves alterations in glucose and fatty acid metabolism. Here, we investigate the role of Carnitine palmitoyl transferase 1a (Cpt1a), a key enzyme in long-chain fatty acid (LCFA) oxidation, in ErbB2-driven breast cancers. In ErbB2+ breast cancer models, ablation of Cpt1a delays tumor onset, growth, and metastasis. However, Cpt1a-deficient cells exhibit increased glucose dependency that enables survival and eventual tumor progression. Consequently, these cells exhibit heightened oxidative stress and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Inhibiting Nrf2 or silencing its expression reduces proliferation and glucose consumption in Cpt1a-deficient cells. Combining the ketogenic diet, composed of LCFAs, or an anti-ErbB2 monoclonal antibody (mAb) with Cpt1a deficiency significantly perturbs tumor growth, enhances apoptosis, and reduces lung metastasis. Using an immunocompetent model, we show that Cpt1a inhibition promotes an antitumor immune microenvironment, thereby enhancing the efficacy of anti-ErbB2 mAbs. Our findings underscore the importance of targeting fatty acid oxidation alongside HER2-targeted therapies to combat resistance in HER2+ breast cancer patients., (© 2024. The Author(s).)

In triple-negative breast cancer (TNBC), stromal restriction of CD8 + T cells associates with poor clinical outcomes and lack of responsiveness to immune-checkpoint blockade (ICB). To identify mediators of T cell stromal restriction, we profiled murine breast tumors lacking the transcription factor Stat3, which is commonly hyperactive in breast cancers and promotes an immunosuppressive tumor microenvironment. Expression of the cytokine Chi3l1 was decreased in Stat3 -/- tumors. CHI3L1 expression was elevated in human TNBCs and other solid tumors exhibiting T cell stromal restriction. Chi3l1 ablation in the polyoma virus middle T (PyMT) breast cancer model generated an anti-tumor immune response and delayed mammary tumor onset. These effects were associated with increased T cell tumor infiltration and improved response to ICB. Mechanistically, Chi3l1 promoted neutrophil recruitment and neutrophil extracellular trap formation, which blocked T cell infiltration. Our findings provide insight into the mechanism underlying stromal restriction of CD8 + T cells and suggest that targeting Chi3l1 may promote anti-tumor immunity in various tumor types., Competing Interests: Declaration of interests J.A.E. is a co-founder of Elkurt Therapeutics and is a founder of and stockholder in and serves on the Scientific Advisory Board for Ocean Biomedical Inc., which develops inhibitors of 18-glycosyl hydrolases as therapeutics. C.G.L. and S.K. are consultants for Ocean Biomedical Inc. J.A.E., C.G.L., and S.K. have a patent entitled “methods and composition relating to anti-CHI3L1 antibody reagents” (US Patent 10253111)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we have shown that deletion of c-Src in a genetically engineered model mimicking the luminal B molecular subtype of breast cancer abrogated the activity of forkhead box M1 (FOXM1), a master transcriptional regulator of the cell cycle. We determined that c-Src phosphorylated FOXM1 on 2 tyrosine residues to stimulate its nuclear localization and target gene expression. These included key regulators of G2/M cell-cycle progression as well as c-Src itself, forming a positive feedback loop that drove proliferation in genetically engineered and patient-derived models of luminal B-like breast cancer. Using genetic approaches and small molecules that destabilize the FOXM1 protein, we found that targeting this mechanism induced G2/M cell-cycle arrest and apoptosis, blocked tumor progression, and impaired metastasis. We identified a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the luminal B subtype, which responds poorly to currently approved therapies. These findings revealed a regulatory network centered on c-Src and FOXM1 that is a targetable vulnerability in aggressive luminal breast cancers.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tumor cells utilize glucose as a primary energy source and require ongoing lipid biosynthesis for growth. Expression of DecR1, an auxiliary enzyme in the fatty acid beta-oxidation pathway, is significantly diminished in numerous spontaneous mammary tumor models and in primary human breast cancer. Moreover, ectopic expression of DecR1 in ErbB2/Neu-induced mammary tumor cells is sufficient to reduce levels of ErbB2/Neu expression and impair mammary tumor outgrowth. This correlates with a decreased proliferative index and reduced rates of de novo fatty acid synthesis in DecR1-expressing breast cancer cells. Although DecR1 expression does not affect glucose uptake in ErbB2/Neu-transformed cells, sustained expression of DecR1 protects mammary tumor cells from apoptotic cell death following glucose withdrawal. Moreover, expression of catalytically impaired DecR1 mutants in Neu-transformed breast cancer cells restored Neu expression levels and increased mammary tumorigenesis in vivo. These results argue that DecR1 is sufficient to limit breast cancer cell proliferation through its ability to limit the extent of oncogene expression and reduce steady-state levels of de novo fatty acid synthesis. Furthermore, DecR1-mediated suppression of tumorigenesis can be uncoupled from its effects on Neu expression. Thus, while downregulation of Neu expression may contribute to DecR1-mediated tumor suppression in certain cell types, this is not an obligate event in all Neu-transformed breast cancer cells.

The displacement of the femoral head along the upper femoral physis that occurs during adolescence or slipped capital femoral epiphysis (SCFE) is not a very common traumatic entity. Ever since Muller1 first described it in 1888, its symptoms, clinical manifestations, diagnosis, treatment and complications have been thoroughly described and studied. Nevertheless little progress has been accomplished as far as its etiology is concerned. In order to assess the potential pathologic influence of any parathyroid hormone (PTH) disturbances on the development of SCFE, we conducted a prospective clinical study with 14 patients, 7 boys and 7 girls (16 hips), suffering from SCFE (Group A). Another 5 patients who had been treated for SCFE a few years before the study, were used as a control group (Group B). We measured the level of I-PTH along with serum calcium (Ca) and phosphorus (P) levels. Furthermore, we checked all the necessary anthropometric characteristics of the patients (i.e., age, height, weight and sexual maturation). Each patient of Group A was categorized from grade I to grade V according to the progress of the slipping. The results showed an increased incidence of serum PTH level abnormalities (both decrease and increase) in Group A while Group B patients had normal results. The detected I-PTH serum level abnormalities were not in any pattern related to the Ca and P serum levels. We believe that a temporary parathyroid hormone disorder during the early years of adolescence may play a potentially significant role (along with other etiologic factors) in the development of SCFE.

It has been previously shown that keratinocytes express a high level of 25-hydroxyvitamin D(3) (25-OHD(3)) 1alpha-hydroxylase (1alpha-hydroxylase). 1alpha-Hydroxylase catalyzes the conversion of 25-OHD(3) to 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3) is both antiproliferative (i.e., suppresses cell growth) and prodifferentiative (i.e., induces cell differentiation) in many cell types. We hypothesized that local production of 1,25(OH)(2)D(3) by keratinocytes may suppress their growth and induce their differentiation in an autocrine fashion. To test this hypothesis, we inactivated both 1alpha-hydroxylase alleles in a ras-transformed keratinocyte cell line, HPK1Aras, which typically produces squamous carcinoma in nude mice. To inactivate 1alpha-hydroxylase expression by HPK1Aras cells, we disrupted both alleles of the 1alpha-hydroxylase gene by homologous recombination. Lack of expression and activity of 1alpha-hydroxylase was confirmed by Northern blot analysis and detected conversion of 25-OHD(3) to 1,25(OH)(2)D(3). We then examined the effect of substrate 25-OHD(3) on parameters of growth and differentiation in the double knockout cell line as compared to wild-type HPK1Aras cells in vitro. It was found that 1alpha-hydroxylase inactivation blocked the antiproliferative and prodifferentiative effect of 25-OHD(3). These in vitro effects were further analyzed in vivo by injecting knockout or control cells subcutaneously in severely compromised immunodeficient mice. Tumor growth was accelerated and differentiation was inhibited in mice given injections of knockout cells as compared to control cells in the presence of substrate 25-OHD(3). Our results demonstrate, for the first time, that 1alpha-hydroxylase expression by keratinocytes plays an important role in autocrine growth and differentiation of these cells, and suggest that expression of this enzyme may modulate tumor growth in squamous carcinomas.

Many human cancers have been associated with the deregulation of receptor tyrosine kinases (RTK). However, the individual contribution of receptor-associated signaling proteins in cellular transformation and metastasis is poorly understood. To examine the role of RTK activated signal transduction pathways to processes involved in cell transformation, we have exploited the oncogenic derivative of the Met RTK (Tpr-Met). Unlike other RTKs, twin tyrosine residues in the carboxy-terminal tail of the Met oncoprotein and receptor are required for all biological and transforming activities, and a mutant lacking these tyrosines is catalytically active but non transforming. Using this mutant we have inserted oligonucleotide cassettes, each encoding a binding site for a specific signaling protein derived from other RTKs. We have generated variant forms of the Tpr-Met oncoprotein with the ability to bind individually to the p85 subunit of PI3'K, PLCgamma, or to the Grb2 or Shc adaptor proteins. Variants that recruit the Shc or Grb2 adaptor proteins generated foci of morphologically transformed fibroblast cells and induced anchorage-independent growth, scattering of epithelial cells and experimental metastasis. In contrast, variants that bind and activate PI3'K or PLCgamma failed to generate readily detectable foci. Although cell lines expressing the PI3'K variant grew in soft-agar, these cells were non metastatic. Using this unique RTK oncoprotein model, we have established that Grb2 or Shc dependent signaling pathways are sufficient for cell transformation and metastatic spread.

We examined parathyroid hormone-related peptide (PTHrP) production and regulation in both normal human melanocytes and in a human amelanotic melanoma cell line (A375). Northern blot and immunocytochemical analysis demonstrated that both cultured A375 cells and normal human melanocytes express PTHrP, but A375 cells expressed much higher levels of the peptide. PTHrP secretory rate increased at least 10-fold after treatment with 10% fetal bovine serum (100.2 +/- 2.8 pmol/10(6) cells vs. basal <15 pmol/10(6) cells) in proliferating A375 cells but only twofold in confluent cells. Treatment of A375 cells with increasing concentrations of 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or its low-calcemic analog EB-1089 revealed that EB-1089 was 10-fold more potent than 1, 25-(OH)(2)D(3) on inhibition of both cell proliferation and PTHrP expression. Furthermore, inoculation of A375 cells into the mammary fat pad of female severe combined immunodeficiency mice resulted in the development of hypercalcemia and elevated concentrations of plasma immunoreactive PTHrP in the absence of detectable skeletal metastases. Our study, therefore, demonstrates a stepwise increase in PTHrP expression when cells progress from normal to malignant phenotype and suggests that EB-1089 should be further evaluated as a therapeutic agent in human melanoma.

1,25-Dihydroxyvitamin D has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of EB 1089, a low calcemic analogue of vitamin D, on the development of osteolytic bone metastases after intracardiac injection of the human breast cancer cell line MDA-MB-231 in nude mice. Animals injected with tumor cells were implanted simultaneously with osmotic minipumps containing either EB 1089 or vehicle. Both groups remained normocalcemic for the duration of the experiment. The total number of bone metastases, the mean surface area of osteolytic lesions, and tumor burden within bone per animal were markedly decreased in EB1089-treated mice. Furthermore, longitudinal analysis revealed that mice treated with EB1089 displayed a marked increase in survival and developed fewer bone lesions and less hind limb paralysis over time as compared with untreated animals. These results suggest that EB1089 may be beneficial in the prevention of metastatic bone lesions associated with human breast cancer.

EB1089, an analogue of 1,25 dihydroxyvitamin D with low calcemic activity is a potent inhibitor of parathyroid hormone-related peptide (PTHRP) production in vitro. The purpose of the present study was to determine whether EB1089 could reverse established hypercalcemia in BALB C nude mice implanted s.c. with a human epithelial cancer previously shown to produce high levels of PTHRP in vitro. Total plasma calcium was monitored before and after tumor development and increased steadily when the tumor reached > or =0.5 cm3. When total calcium was 22.85 mmol/liter, animals were treated with a constant infusion of EB1089 or vehicle alone for a period of 2 weeks. A significant and sustained reduction of plasma calcium from 3.2+/-0.1 to 2.7+/-0.08 (P < 0.01) mmol/liter was observed during infusion with EB1089. In contrast, calcium levels in vehicle-treated animals continued to rise during the infusion period. Tumor growth velocity also slowed significantly after the administration of EB1089 as compared with vehicle-treated animals. Plasma PTHRP levels measured at the end of the 2 weeks' infusion period were significantly lower in animals treated with EB1089 as compared with animals treated with vehicle alone (44+/-8 pg/ml versus 194+/-35 pg/ml, P < 0.001). These results, therefore, demonstrate that EB1089 can reverse established hypercalcemia in a human model of squamous cancer.

Tuberculosis with secondary staphylococcal infection destroyed the right thumb and index phalanges of a 24-year-old farmer. Four months after starting antibiotic therapy the index finger was amputated, and the thumb reconstructed by excising the disorganized phalanges and transferring the index metacarpal as a free graft to replace them. No previous report of this technique has been traced. A concurrent infection of the patient's left tarsus was treated by triple arthrodesis.