The tumor-derived cytokine Chi3l1 induces neutrophil extracellular traps that promote T cell exclusion in triple-negative breast cancer.

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.

Nanoliposome targeting in breast cancer is influenced by the tumor microenvironment.

MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.

Coordinated activation of c-Src and FOXM1 drives tumor cell proliferation and breast cancer progression.

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.

HER2Δ16 Engages ENPP1 to Promote an Immune-Cold Microenvironment in Breast Cancer.

The tumor-immune microenvironment (TIME) is a critical determinant of therapeutic response. However, the mechanisms regulating its modulation are not fully understood. HER2Δ16, an oncogenic splice variant of the HER2, has been implicated in breast cancer and other tumor types as a driver of tumorigenesis and metastasis. Nevertheless, the underlying mechanisms of HER2Δ16-mediated oncogenicity remain poorly understood. Here, we show that HER2∆16 expression is not exclusive to the clinically HER2+ subtype and associates with a poor clinical outcome in breast cancer. To understand how HER2 variants modulated the tumor microenvironment, we generated transgenic mouse models expressing either proto-oncogenic HER2 or HER2Δ16 in the mammary epithelium. We found that HER2∆16 tumors were immune cold, characterized by low immune infiltrate and an altered cytokine profile. Using an epithelial cell surface proteomic approach, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as a functional regulator of the immune cold microenvironment. We generated a knock-in model of HER2Δ16 under the endogenous promoter to understand the role of Enpp1 in aggressive HER2+ breast cancer. Knockdown of Enpp1 in HER2Δ16-derived tumor cells resulted in decreased tumor growth, which correlated with increased T-cell infiltration. These findings suggest that HER2Δ16-dependent Enpp1 activation associates with aggressive HER2+ breast cancer through its immune modulatory function. Our study provides a better understanding of the mechanisms underlying HER2Δ16-mediated oncogenicity and highlights ENPP1 as a potential therapeutic target in aggressive HER2+ breast cancer.

Elevated expression of DecR1 impairs ErbB2/Neu-induced mammary tumor development.

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.

An ErbB2/c-Src axis links bioenergetics with PRC2 translation to drive epigenetic reprogramming and mammary tumorigenesis.

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.

Translational and HIF-1α-Dependent Metabolic Reprogramming Underpin Metabolic Plasticity and Responses to Kinase Inhibitors and Biguanides.

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.

Use of signal specific receptor tyrosine kinase oncoproteins reveals that pathways downstream from Grb2 or Shc are sufficient for cell transformation and metastasis.

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.

Targeted disruption of the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in ras-transformed keratinocytes demonstrates that locally produced 1alpha,25-dihydroxyvitamin D3 suppresses growth and induces differentiation in an autocrine fashion.

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.

Elongation of the femoral neck in Perthes disease.

We present the long-term clinical and radiological results of a series of 168 young patients with unilateral Perthes disease who were treated in our department between 1989 and 1997, using a combined osteotomy in the longitudinal and horizontal axis of the proximal femur and elongation of the femoral neck. Surgical treatment was undertaken for any group II (Catterall's classification) patient, with the presence of two or more radiographic signs of the "head at risk" and the clinical sign of flexion with abduction, as well as for all cases classified by Catterall as groups III and IV. The surgical procedure we describe provided 147 radiologically and clinically normal hips in the short and long-term. However, in the long-term, 21 out of 168 patients presented with residual deformities such as shallow acetabulum, thickening of the acetabular floor, coxa magna, thicker and slightly shorter femoral neck. Thus for the vast majority of patients, the operation we describe here provided leg length equalisation and restored the working length of the abductors by maintaining the tip of the greater trochanter at the same level as on the unaffected side.

Parathyroid hormone-related peptide and survival of patients with cancer and hypercalcemia.

PURPOSE: Parathyroid hormone-related peptide (PTHrP) is the predominant cause of malignancy-associated hypercalcemia. However, its prognostic utility is unclear. We aimed to determine the prognostic value of serum PTHrP levels in patients who had hypercalcemia associated with malignancy. METHODS: In this prospective case series, we evaluated 76 patients with a diagnosis of cancer and hypercalcemia (serum calcium level >/=10.3 mg/dL on at least two occasions). PTHrP levels >/=1 pmol/L were considered elevated. We used multivariate Cox regression analysis to identify factors associated with mortality. RESULTS: Fifty patients (66%) died during follow-up. In a multivariate analysis, higher pretreatment calcium levels and elevated PTHrP levels were associated with increased mortality, with effects of PTHrP varying by age (P = 0.03). Survival was associated with pretreatment calcium levels both in patients over 65 years of age (hazard ratio [HR] per mg/dL = 1.5; 95% confidence interval [CI]: 1.2 to 1.8; P <0.001) and in patients aged 65 years or less (HR = 1.3; 95% CI: 1.1 to 1.5; P = 0.003). Adjusted for pretreatment calcium levels, elevated PTHrP levels were associated with increased mortality in patients aged

Pamidronate prevents the development of skeletal metastasis in nude mice transplanted with human breast cancer cells by reducing tumor burden within bone.

Pamidronate is used routinely in the treatment of established bone metastasis. However, pamidronate has not yet been assessed in the prevention of osteolytic bone metastasis and its precise mechanism of action in this disorder remains to be established. In the present study, pamidronate or vehicle alone was administered subcutaneously to nude mice either simultaneously or as post intracardiac injection of the human breast cancer MDA-MB-231 cells. Radiographs were used first to assess the presence of osteolytic bone metastases. Kaplan-Meier analysis demonstrated that animals treated with pamidronate early, but not late, showed a slower progression of bone metastases and hind limb paralysis than did vehicle-treated animals. Mann-Whitney analysis showed that only 44.4% of mice treated with pamidronate at the time of tumor cell inoculation developed bone metastases as compared to over 80% (p<0.05) of mice receiving vehicle alone. We then analyzed the number of bone lesions and their volume at time of sacrifice by bone histomorphometry. In contrast to X-ray analysis, morphometric analysis indicates that the number of lesions within bone was similar in pamidronate and vehicle-treated mice but that the lesions were significantly smaller and therefore, often not visible on radiographs. These results demonstrate that pamidronate is effective in reducing tumor burden in breast cancer metastatic to bone and is most effective as a preventative agent when administered closest in time to implantation of tumor cells. Our data also suggest that pamidronate acts mainly by inhibiting the growth of established bone metastatic lesions but has no effect on the metastatic spread itself.

A novel surgical option for the operative treatment of clubfoot.

Clubfoot (talipes equinovarus) is a condition well known since the time of Hippocrates. Numerous conservative treatments have been introduced for this condition; few are still in favour. Conservative treatment was used in our department up to the third month of age. The indication for surgery was failure to correct or maintain the correction after conservative treatment. We report on 134 children (206 feet) who had operative treatment for clubfoot in our department during the period 1990-1996, using a novel surgical technique based on extensive posteromedial release combined with the lateral spread of the "extensor forces" of the foot. This new technique has produced excellent results.

Talus fracture associated with a fracture dislocation of the distal tibia in an immature skeleton.

A 12-year-old boy with a severe injury of his right ankle was treated in the accident unit. The local condition was an extensive open wound with parts of the fractured bones prominent. Radiographs showed a severely displaced fracture of the body of the talus associated with a Salter-Harris IV injury of the distal tibia and subluxation of the ankle. After proper debridement the fractured bones were reduced and fixed. Eleven years later both fractures are completely healed without any sign of avascular necrosis, the patient has full painless ankle movement and he can perform his demanding occupation without any problem.

Surgical treatment of developmental dysplasia of the hip in the periadolescent period.

The surgical management of patients with neglected developmental dysplasia of the hip (DDH) after the age of 6 years has been the subject of controversy. We present 11 cases (16 hips) of neglected DDH that were treated operatively by means of open reduction and derotational subtrochanteric osteotomy. Patient age ranged between 10 and 17 years (mean, 12 years). Follow-up ranged from 5 to 13 years (mean, 8.7 years). The results have been satisfactory both clinically (evaluated using the modified Harris hip score) and radiographically (evaluated using Severin's classification). Our data suggest that neglected DDH cases, not only during early childhood, but also in the periadolescent period, should be considered for surgical treatment.

Outcome of physeal and epiphyseal injuries of the distal tibia with intra-articular involvement.

The authors reviewed 83 physeal and epiphyseal injuries of the distal tibia with intra-articular involvement. The children, aged 11 to 14 years, were treated in the authors' department during 1987 to 1999. Treatment was nonoperative for 72.25% (60/83) and surgical for 27.75% (23/83) according to specific indications. This gives the basis for a classification of these injuries. The main purpose of the study was to investigate the long-term results of these injuries according to a radiologic classification. The parameters considered were the patient's age, the mechanism of injury, and the possibility of growth deformities or functional disorders. They were studied relative to the long-term results, with a follow-up of 2 to 13 years. Regardless of treatment, varus deformity, ranging from 10 to 15 degrees in relation to the normal opposite leg, occurred in four cases. In only one case was there painful limitation of ankle joint movement; in two other children an overgrowth of the medial malleolus was detected, with no functional impairment.

Posteromedial dislocation of the elbow with lateral condyle fracture in children.

Posteromedial dislocation of the elbow in children is an extremely rare injury. The current study describes four boys with posteromedial dislocation of the elbow associated with a displaced fracture of the lateral humeral condyle. The patients ranged in age from 6 to 12 years (average age, 9 years 6 months). Closed reduction of the elbow dislocation and open reduction of the lateral humeral condyle fracture with fixation by Kirschner wires by a lateral approach was done in each patient. This was followed by 4 weeks immobilization wearing a long-arm cast. There were three excellent results and one good result. Healing and return of normal function occurred in all but one patient who had minor loss of full extension. Average duration of followup was 7 years 6 months (range, 3-13 years). Dislocation of the elbow associated with a displaced fracture of the lateral humeral condyle can be treated by closed reduction of the elbow dislocation and open anatomic reduction and fixation of the lateral condyle fracture with good results.

Rotational injuries of the distal tibial growth plate.

Nine rotational injuries of the distal tibial growth plate in combination with spiral fracture of the fibula were treated in our department between 1993 and 2000. The average age of the patients was 12 years. The injury was a result of sudden, forceful external rotation of the ankle and foot. Pain and slight swelling of the ankle and external rotation of the distal tibia of 20 degrees -40 degrees were present. Radiologically, irregularity and widening of the growth plate of the distal tibia were obvious. In all these cases we noted, on lateral views, the characteristic sign of an "open fish mouth," due to the changes in the shape of the physeal plate. The spiral fracture of the fibula was not obvious in all radiographic views. The treatment in all eight fresh cases was closed reduction and manipulation by reversing the mechanism of injury and long leg cast application in all nine cases (including the one neglected case) for 6-8 weeks. The final results were either excellent or very good in all nine patients after a mean follow-up of 6 years.