Development of a contrast-enhanced ultrasound guided high intensity focused ultrasound system for coagulation of liver parenchyma.

BACKGROUND: The liver is the most common organ injured in blunt abdominal trauma and makes up roughly 5% of all trauma admissions. Current treatments are invasive and resource-intensive, which may delay care. We aim to develop and validate a contrast-enhanced ultrasound (CEUS)guided noninvasive tool to treat liver lacerations at the bedside. METHODS: Two 1.8 MHz high-intensity focused ultrasound (HIFU) elements were coupled to a C1-6 diagnostic ultrasound probe and a Logiq E10 scanner (GE HealthCare) utilizing a custom enclosure for co-registered imaging and ablation. A phantom was created from polyacrylamide gel combined with thermochromic ink whose color changes above biological ablative temperatures (60 °C). The HIFU wave was focused approximately 0.5 cm below the surface utilizing a 50% duty cycle generating 11.9 MPa for 20, 30, 40, 50, and 60s. Experiments were repeated on ex vivo chicken livers in a water bath. Finally, the livers of 4 live swine underwent up to 6 CEUS-guided treatments using parameters optimized from in vitro work. RESULTS: Treatment of the phantom between 20-60s, produced ablation sizes from 0.016 to 0.4 cm 3 . The relationship between time and size was exponential (R 2 = 0.992). Ablation areas were also well visualized on with ultrasound imaging. The ex vivo liver ablation size at 20s was 0.37 cm 3 , at 30s was 0.66 cm 3 , and at 100 s was 5.0 cm 3 . For the in-vivo swine experiments, the average ablation area measured 2.0x0.75 cm with a maximum of 3.5x1.5 cm. CEUS was utilized with the contrast agent Definity (Lantheus) for identification of lacerations as well as immediate post operative evaluation of therapy. CONCLUSION: These experiments demonstrate the feasibility of CEUS guided transdermal HIFU ablation and the time-dependent size of ablation. This work warrants future investigations into using ultrasound to detect active bleeding and HIFU to coagulate grade III and IV liver laceration. STUDY TYPE: Therapeutic/care management.

Challenges in the Management of Symptomatic Fallopian Canal Meningoceles: A Multicenter Case Series and Literature Review.

OBJECTIVE: To describe the presentations, the diagnosis, our treatment approaches, and the outcomes for 11 patients with fallopian canal meningocele (FCM). STUDY DESIGN MULTICENTER: Retrospective case series. SETTING: Tertiary referral centers. PATIENTS: Patients (N = 11) with radiographically or intraoperatively identified, symptomatic FCM. INTERVENTIONS: Surgical repair of cerebrospinal fluid (CSF) leak and meningocele versus observation. MAIN OUTCOME MEASURES: Presentation (including symptoms, radiographic imaging, and comorbidities), management (including surgical approach, technique for packing, use of lumbar drain), clinical outcomes (control of CSF leak, meningitis, facial nerve function), and revision surgery. RESULTS: Patients presented with spontaneous CSF leak (n = 7), conductive (N = 11) and sensorineural hearing loss (n = 3), nonpositional intermittent vertigo (n = 3), headaches (n = 4), and recurrent meningitis (n = 1). Risk factors in our series included obesity (n = 4), Chiari 1 malformation (n = 1), and head trauma (n = 2). Noncontrast computed tomography of the temporal bone and magnetic resonance imaging were positive for FCM in 10 patients. Eight patients were managed surgically via a transmastoid approach (n = 4), combined transmastoid and middle fossa (N = 3), or middle fossa alone (n = 1); three were managed conservatively with observation. Postoperative complications included worsened facial nerve palsy (n = 1), recurrent meningitis (n = 1), and persistent CSF leak that necessitated revision (n = 1). CONCLUSIONS: Facial nerve meningoceles are rare with variable presentation, often including CSF otorrhea. Management can be challenging and guided by symptomatology and comorbidities. Risk factors for FCM include obesity and head trauma, and Chiari 1 malformation may present with nonspecific otologic symptoms, in some cases, meningitis and facial palsy. Layered surgical repair leads to high rates of success; however, this may be complicated by worsening facial palsy.

Presence of 10 transformation zone cells in endocervical curettage should not be required for adequacy.

OBJECTIVES: In another publication, we concluded endocervical curettage (ECC) should have a minimum number of squamous cells for adequacy, similar to the requirements for adequate cervical Papanicolaou smears. Here, we investigate if also, similar to cervical Papanicolaou smears, the presence of at least 10 cells from the endocervical/transformation zone (EC/TZ) in ECCs should be used as a quality assurance measure or if, instead, at least 10 EC/TZ cells should be part of the adequacy criteria for ECC, with an emphasis on diagnosis of at least high-grade squamous dysplasia (HGD). METHODS: All patients with at least HGD diagnosed on an excisional biopsy specimen (loop electrosurgical excision procedure [LEEP]) from May 1, 2018, to December 31, 2019, and an ECC in the preceding 6 months at our institution were included. Number of EC/TZ cells present in ECCs was counted visually and categorized as less than or greater than 10 TZ cells. A χ2 test was used to evaluate the proportion of ECCs with and without HGD and the presence or absence of at least 10 EC/TZ cells. Given our recent work encouraging at least 1000 squamous cells in an ECC to be considered adequate, we also evaluated only ECCs with greater than 1000 squamous cells with and without HGD and the presence or absence of at least 10 EC/TZ cells. P value was <.05. RESULTS: Fifty-one LEEPs with HGD and a preceding ECC in the previous 6 months were identified. Of the 51 ECCs, 6 had fewer than 10 EC/TZ cells and 45 had at least 10 EC/TZ cells. A similar proportion of the ECCs with HGD had at least 10 EC/TZ cells as those without HGD (93% vs 86%, P = .53). Using only ECCs with greater than 1000 squamous cells, we still found no statistical difference in the proportion of ECCs with HGD having greater than 10 EC/TZ cells compared to those without HGD (91% vs 100%, P = .49). CONCLUSIONS: We found that the presence of at least 10 EC/TZ cells does not increase the likelihood of finding HGD in an ECC performed in the 6 months prior to a LEEP with HGD. Similar to the use of the TZ component in cervical Papanicolaou smears, the presence or absence of at least 10 TZ cells in an ECC should only be considered a quality assurance measure and not be used as a criterion for adequacy of the specimen.

Telecytology rapid onsite evaluation, with real-time communication between cytopathologist, cytotechnologist, and proceduralist, offers better adequacy rates for lymph node, but not thyroid, fine-needle aspirations.

INTRODUCTION: Rapid onsite evaluation (ROSE) decreases rates of inadequate fine-needle aspirations (FNAs). Telecytology allows pathologists to perform ROSE without being physically "on site", thereby saving cytopathologists' travel time and allowing them to perform ROSE for multiple institutions. Little research exists comparing telecytology to non-telecytology ROSE for FNA inadequacy rates. MATERIALS AND METHODS: Using previously obtained quality metrics, we compared inadequacy rates for lymph node and thyroid FNAs with and without ROSE and with non-telecytology ROSE compared with telecytology ROSE. Use of ROSE was determined by the proceduralist. Type of ROSE was location-based, as only certain locations at our institution have telecytology capabilities. Chi-squared testing was used to compare proportions of populations and P value was set to 0.05. RESULTS: A total of 1168 lymph node and 1177 thyroid FNAs were included in our adequacy analysis. We found any ROSE decreased our inadequacy rate for both lymph node (20.4% to 12.7%, P = 0.002) and thyroid (34.7% to 4.8%, P = 7.4 × 10-18) FNAs. We found telecytology further decreased our inadequacy rate for lymph node (13.8% to 5.9%, P = 0.016), but not thyroid (3.3% to 5.0%, P = 0.34), FNAs. CONCLUSIONS: At our institution, when using telecytology, slides are read in real time with the cytotechnologist and the proceduralist looking at slides together near the patient bedside, while the cytopathologist is on the phone looking at slides on the computer screen via Dameware. When non-telecytology ROSE is performed, the cytotechnologist evaluates a slide, brings it to the cytopathologist's office and then the cytopathologist calls the proceduralist to discuss the slide. We believe telecytology offers an opportunity for more inclusive communication thereby improving adequacy rates for more complex cases, like lymph nodes, without affecting adequacy rates for cases where assessment of adequacy is less complex, like thyroid. This research supports use of telecytology especially for complex cases.

Cytology-histology correlation of cervical Papanicolaou smears and biopsies performed at a single institution compared to those performed at different institutions.

OBJECTIVE: Cytology-histology correlation (CHC) is the gold standard of quality assurance in cytology laboratories to ensure appropriate patient treatment, and as an educational tool for cytology laboratory personnel. If cervical Pap smears (CPs) and cervical biopsies (CBs) are performed at different institutions, these benefits may be lost. METHODS: All CBs performed at our institution from 1 January 2019 to 31 December 2019 with adequate CPs performed in the 6 months prior to the CB were included in this retrospective review. We compared the CHC for CPs and CBs performed at a single institution to the CHC for CPs and CBs performed at different institutions, with a focus on the proportion of overcalls on CPs, as those are the most challenging discrepant CHC to manage clinically. We used the American Society of Cytology guidelines for our discrepancy assessment grid. A Chi-squared test was used to compare the proportions of the populations. The P-value was set at < 0.05. RESULTS: Of the 305 CBs in our study population, 69 had a CP performed at our institution and 236 had a CP performed at an outside institution. The CHC for CBs and CPs performed at a single institution showed statistically significantly less disagreement than the CHC for those performed at different institutions (P < 0.05). Further, CBs and CPs performed at a single institution had statistically significantly fewer overcalls than CBs and CPs performed at different institutions (P < 0.05). CONCLUSION: This study further supports the use of CHC, and in light of our findings we recommend that a patient's CPs and CBs are performed at the same institution. If performing a CP and CB at the same institution is not feasible, a prospective consultation review of the CP by the institution performing the CB should be strongly considered. Further study, including an evaluation of the reason for the discrepancy in discordant cases may better elucidate the reasons for better CHC agreement when CP and CB are performed at the same institution.

Adequacy in Endocervical Curettage.

OBJECTIVES: Specimen adequacy is an important quality assurance component of a cervical Papanicolaou test. Although consensus exists on minimal acceptable cellularity for cervical Papanicolaou tests, no such criteria exist for endocervical curettage (ECC) specimens. We sought to identify minimum acceptable cellularity for accurate diagnosis of high-grade dysplasia (HGD) on ECC. METHODS: All patients with HGD diagnosed in a loop electrosurgical excision procedure (LEEP) from May 8, 2018, to December 18, 2019, and an ECC in the preceding 6 months at our institution were included (n = 51). All ECCs performed before the LEEP were evaluated for cellularity of squamous cells using Aperio eSlide Manager (Leica Biosystems). Biopsy results concurrent with the ECC were noted. We compared the number of squamous cells in positive and negative ECC specimens using a t-test. The proportion of ECC specimens and concurrent biopsies undergoing immunohistochemical (IHC) staining for p16 were compared using the χ2 test. P < .05 was considered significant. RESULTS: Endocervical curettage specimens positive for HGD have increased cellularity compared with negative ECC specimens (mean cellularity, 10,165 vs 1,055; P < .05). Further, IHC staining for p16 was more likely to be performed on an ECC specimen positive for HGD than on a negative ECC specimen (50% vs 3%; P < .05). Biopsies performed concurrently with a negative ECC finding were more likely to undergo p16 IHC than biopsies performed concurrently with a positive ECC finding (51% vs 7%; P < .05). Finally, we observed no difference in the proportion of biopsies undergoing IHC staining for p16 when comparing biopsies positive for HGD with negative biopsies (37% vs 46%; P = .33). CONCLUSIONS: We find cellularity of approximately 10,000 cells adequate to diagnose HGD in an ECC specimen and cellularity of approximately 1,000 cells to be inadequate. Further, we find p16 IHC commonly used as a "rule-in" test on ECC specimens at our institution. Biopsies accompanying an ECC specimen negative for HGD are more likely to undergo p16 IHC than those accompanying an ECC specimen positive for HGD, but there is no difference in the proportion of biopsies undergoing p16 IHC when comparing positive and negative results in the biopsies themselves. These findings further support the need for adequate cellularity for diagnosis in ECC, especially when a biopsy is technically difficult. Further areas for exploration include investigating laboratory procedures to maximize the cellularity of ECC specimens.

Rare occurrence of pseudomyxoma peritonei (PMP) syndrome arising from a malignant transformed ovarian primary mature cystic teratoma treated by cytoreductive surgery and HIPEC: a case report.

BACKGROUND: Pseudomyxoma peritonei (PMP) syndrome is a disease process that typically occurs from ruptured appendiceal mucocele neoplasms. PMP syndrome arising from malignant transformation of an ovarian primary mature cystic teratoma (MCT) is a pathogenesis rarely encountered. CASE PRESENTATION: Herein, we report a 28-year-old patient evaluated and treated for a right ovarian mass and large volume symptomatic abdominopelvic mucinous ascites. Molecular profiling and genetic analysis revealed mutations in ATM, GNAS, and KRAS proteins while IHC demonstrated gastrointestinal-specific staining for CK20, CDX2, CK7, and SATB2. Peritoneal cytology showed paucicellular mucin. Diffuse peritoneal adenomucinosis (DPAM) variant of PMP arising from a ruptured ovarian primary MCT after malignant transformation to a low-grade appendiceal-like mucinous neoplasm was ultimately confirmed. Treatment included staged therapeutic tumor debulking and right salpingo-oophorectomy followed by cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC). CONCLUSIONS: Our report builds upon the existing literature supporting this aggressive treatment option reserved for advanced abdominal malignancies utilized in this patient with a rare clinical entity.

Primary cervical ganglioneuroblastoma, nodular subtype.

Primary cervical ganglioneuroblastoma is rare and reports of its subtypes are limited. This case series describes two pediatric patients with the nodular subtype of primary cervical ganglioneuroblastoma with lymphatic spread. Clinical course, diagnosis, and management of this rare tumor are discussed with emphasis on the importance of including neuroblastic tumors in the differential diagnosis of pediatric neck masses. We also report the use of nerve monitoring of the recurrent laryngeal nerve as a surrogate for the vagus nerve during a pediatric neck dissection.

Mitotic count of fine needle aspiration material of gastrointestinal stromal tumours of the stomach underestimates actual mitotic count.

INTRODUCTION: A mitotic count is required for histological grading in resections of gastrointestinal stromal tumours (GISTs). However, no consensus on the utility of mitotic count in fine needle aspiration (FNA) GIST material currently exists. This study examines the relationship between mitotic counts of FNAs and subsequent resections of GISTs of the stomach. MATERIALS AND METHODS: We identified 39 cases of GISTs of the stomach diagnosed via FNA at our institution between January 1, 2014, and December 31, 2019, with subsequent resection. We noted if rapid on-site evaluation (ROSE) was performed. Cell block (CB) material from FNAs was analysed for total area, percentage containing neoplastic cells, and number of mitoses. We compared the mitotic counts in CBs and subsequent resections with t tests. RESULTS: ROSE was performed in 82% of cases and called adequate every time. Mean values for total CB area, neoplastic material percentage, and area of neoplastic cells were 54.7 mm2 (range 1-986), 45% (range 10%-90%), and 19.2 mm2 , respectively; 27 cases (69%) had greater than 50 high powered fields of GIST material in the CB. Mean numbers of mitoses per 5 mm2 were 0.38 (range 0-11) for CBs versus 5.92 (range 0-70) for resections (P < 0.05). CONCLUSION: At our institution, ROSE adequacy of spindle cell lesions focuses on diagnosing GIST rather than obtaining adequate material for histological grading. Mitotic figures were statistically lower in FNA CB material than subsequent resections, and using mitotic counts from CB material may underestimate the histological grade of GISTs of the stomach.

Microcystic, Elongated, and Fragmented Pattern Invasion in Ovarian Endometrioid Carcinoma: Immunohistochemical Profile and Prognostic Implications.

Microcystic, elongated, and fragmented (MELF) pattern invasion is a poor prognostic indicator in uterine endometrioid carcinoma, but its existence, biology, and prognostic value have not been described in ovarian endometrioid carcinoma. We evaluated cases of ovarian endometrioid carcinoma without synchronous uterine endometrioid carcinoma for MELF and other histologic features. To evaluate tumor biology, we assessed an immunohistochemical profile, including MLH1, PMS2, MSH2, MSH6, ß-catenin, e-cadherin, CK19, and cyclin D1. A retrospective chart review evaluated clinical and demographic features and survival. The Fisher exact test analyzed data. The Kaplan-Meier method assessed overall survival. Forty-two patients met inclusion criteria. MELF was found in 45%. Two MELF cases showed MSH2/MSH6 deficiency and 2 conventional cases showed PMS2 deficiency. Clear cell features were seen exclusively in MELF cases (P-value=0.044). No difference was identified in overall survival, cancer recurrence, serous features, concurrent endometriosis, lymphovascular space invasion, lymph node metastasis, bilaterality of disease, extranodal metastasis, or remainder of immunohistochemical profile. MELF occurs at similar rates in ovarian endometrioid carcinoma and uterine endometrioid carcinoma and can be helpful in defining ovarian endometrioid carcinoma as it proves definitive invasion. Recurrence and overall survival in ovarian endometrioid carcinoma are not affected by MELF. Clear cell features are identified exclusively in MELF cases. Different mismatch repair proteins are lost in MELF compared with conventional ovarian endometrioid carcinomas. Given its association with clear cell features and mismatch repair protein loss, presence of MELF may be useful in clinical decisions regarding surgical staging and Lynch syndrome screening.

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas In Vivo and Ex Vivo.

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K-mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice variant-expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. Mol Cancer Ther; 17(1); 84-95. ©2017 AACR.

Identification of a Substrate-selective Exosite within the Metalloproteinase Anthrax Lethal Factor.

The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.

An Initiative to Standardize the Identification of and Acute Response to Postoperative Lower-Extremity Neurological Deficits: Effects on Provider Knowledge, Confidence, and Communication Skills.

BACKGROUND: Although relatively uncommon, neurological deficits following hip and knee arthroplasty can have permanent and debilitating consequences. This study was conducted to quantify the effectiveness of an educational curriculum aimed at standardizing the identification of and acute response to postoperative neurological deficits in the inpatient setting, specifically with respect to improvements in clinician knowledge, confidence levels, and communication skills. METHODS: A multidisciplinary committee at a single, high-volume academic institution created an algorithm delineating the appropriate clinical actions and escalation procedures in the setting of a postoperative neurological deficit for each clinical practitioner involved in care for patients who undergo arthroplasty. An educational curriculum composed of online learning modules and an in-person "boot camp" featuring simulations with standardized patients was developed, along with assessments of clinician knowledge, confidence levels, and communication skills. Nurses, physical therapists, physician assistants, residents, fellows, and attending surgeons were encouraged to participate. The intervention spanned a 5-month period in 2015 with a mean time of 18.4 weeks between baseline assessments and the time of the latest follow-up. RESULTS: Online modules were completed by 322 individuals, boot camp was completed by 70 individuals, and latest assessments were completed by 38 individuals. The percentage correct on the knowledge assessment increased from 74.5% before the learning modules to 89.5% immediately after (p < 0.001) but degraded over time such that there was no significant difference between baseline and the latest follow-up scores (p = 0.11). Over the course of the boot camp, physician assistants and residents successfully performed approximately 91% of the indicated actions on the scoring rubric; physical therapists and nurses successfully performed 78%. Scores on the communication skills assessment showed a significant mean increase (p = 0.02) over the course of the intervention from 30.32 to 32.50, and the mean self-assessed confidence survey scores increased by 16.7%, from 7.2 to 8.4 (p < 0.001). CONCLUSIONS: A multimodality educational curriculum aimed at quality improvement can produce significant knowledge improvements, but these gains may not be maintained over time without further instruction. Gains in confidence and communication skills appear to be more long-lasting.

An In Vivo Reporter to Quantitatively and Temporally Analyze the Effects of CDK4/6 Inhibitor-Based Therapies in Melanoma.

Aberrant cell-cycle progression is a hallmark feature of cancer cells. Cyclin-dependent kinases 4 and 6 (CDK4/6) drive progression through the G1 stage of the cell cycle, at least in part, by inactivating the tumor suppressor, retinoblastoma. CDK4/6 are targetable and the selective CDK4/6 inhibitor, palbociclib, was recently FDA approved for the treatment of estrogen receptor-positive, HER2-negative advanced breast cancer. In cutaneous melanoma, driver mutations in NRAS and BRAF promote CDK4/6 activation, suggesting that inhibitors such as palbociclib are likely to provide therapeutic benefit in combination with BRAF inhibitors and/or MEK inhibitors that are FDA-approved. However, the determinants of the response to CDK4/6 inhibitors alone and in combination with other targeted inhibitors are poorly defined. Furthermore, in vivo systems to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and determine the extent that CDK activity is reactivated during acquired resistance are lacking. Here, we describe the heterogeneous effects of CDK4/6 inhibitors, the expression of antiapoptotic proteins that associate with response to CDK4/6 and MEK inhibitors, and the development of a luciferase-based reporter system to determine the effects of CDK4/6 inhibitors alone and in combination with MEK inhibitors in melanoma xenografts. These findings are likely to inform on-going and future clinical trials utilizing CDK4/6 inhibitors in cutaneous melanoma. Cancer Res; 76(18); 5455-66. ©2016 AACR.

Identification of exosite-targeting inhibitors of anthrax lethal factor by high-throughput screening.

Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.

Autophagy and senescence in cancer-associated fibroblasts metabolically supports tumor growth and metastasis via glycolysis and ketone production.

Senescent fibroblasts are known to promote tumor growth. However, the exact mechanism remains largely unknown. An important clue comes from recent studies linking autophagy with the onset of senescence. Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST). To test this hypothesis, human fibroblasts immortalized with telomerase (hTERT-BJ1) were stably transfected with autophagy genes (BNIP3, CTSB or ATG16L1). Their overexpression was sufficient to induce a constitutive autophagic phenotype, with features of mitophagy, mitochondrial dysfunction and a shift toward aerobic glycolysis, resulting in L-lactate and ketone body production. Autophagic fibroblasts also showed features of senescence, with increased p21(WAF1/CIP1), a CDK inhibitor, cellular hypertrophy and increased ß-galactosidase activity. Thus, we genetically validated the existence of the autophagy-senescence transition. Importantly, autophagic-senescent fibroblasts promoted tumor growth and metastasis, when co-injected with human breast cancer cells, independently of angiogenesis. Autophagic-senescent fibroblasts stimulated mitochondrial metabolism in adjacent cancer cells, when the two cell types were co-cultured, as visualized by MitoTracker staining. In particular, autophagic ATG16L1 fibroblasts, which produced large amounts of ketone bodies (3-hydroxy-butyrate), had the strongest effects and promoted metastasis by up to 11-fold. Conversely, expression of ATG16L1 in epithelial cancer cells inhibited tumor growth, indicating that the effects of autophagy are compartment-specific. Thus, autophagic-senescent fibroblasts metabolically promote tumor growth and metastasis, by paracrine production of high-energy mitochondrial fuels. Our current studies provide genetic support for the importance of "two-compartment tumor metabolism" in driving tumor growth and metastasis via a simple energy transfer mechanism. Finally, ß-galactosidase, a known lysosomal enzyme and biomarker of senescence, was localized to the tumor stroma in human breast cancer tissues, providing in vivo support for our hypothesis. Bioinformatic analysis of genome-wide transcriptional profiles from tumor stroma, isolated from human breast cancers, also validated the onset of an autophagy-senescence transition. Taken together, these studies establish a new functional link between host aging, autophagy, the tumor microenvironment and cancer metabolism.

Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells.

Here, we show that tamoxifen resistance is induced by cancer-associated fibroblasts (CAFs). Coculture of estrogen receptor positive (ER+) MCF7 cells with fibroblasts induces tamoxifen and fulvestrant resistance with 4.4 and 2.5-fold reductions, respectively, in apoptosis compared with homotypic MCF7 cell cultures. Treatment of MCF7 cells cultured alone with high-energy mitochondrial "fuels" (L-lactate or ketone bodies) is sufficient to confer tamoxifen resistance, mimicking the effects of coculture with fibroblasts. To further demonstrate that epithelial cancer cell mitochondrial activity is the origin of tamoxifen resistance, we employed complementary pharmacological and genetic approaches. First, we studied the effects of two mitochondrial "poisons," namely metformin and arsenic trioxide (ATO), on fibroblast-induced tamoxifen resistance. We show here that treatment with metformin or ATO overcomes fibroblast-induced tamoxifen resistance in MCF7 cells. Treatment with the combination of tamoxifen plus metformin or ATO leads to increases in glucose uptake in MCF7 cells, reflecting metabolic uncoupling between epithelial cancer cells and fibroblasts. In coculture, tamoxifen induces the upregulation of TIGAR (TP53-induced glycolysis and apoptosis regulator), a p53 regulated gene that simultaneously inhibits glycolysis, autophagy and apoptosis and reduces ROS generation, thereby promoting oxidative mitochondrial metabolism. To genetically mimic the effects of coculture, we next recombinantly overexpressed TIGAR in MCF7 cells. Remarkably, TIGAR overexpression protects epithelial cancer cells from tamoxifen-induced apoptosis, providing genetic evidence that increased mitochondrial function confers tamoxifen resistance. Finally, CAFs also protect MCF7 cells against apoptosis induced by other anticancer agents, such as the topoisomerase inhibitor doxorubicin (adriamycin) and the PARP-1 inhibitor ABT-888. These results suggest that the tumor microenvironment may be a general mechanism for conferring drug resistance. In summary, we have discovered that mitochondrial activity in epithelial cancer cells drives tamoxifen resistance in breast cancer and that mitochondrial "poisons" are able to re-sensitize these cancer cells to tamoxifen. In this context, TIGAR may be a key "druggable" target for preventing drug resistance in cancer cells, as it protects cancer cells against the onset of stress-induced mitochondrial dys-function and aerobic glycolysis.