Natalya Chernichenko

GFRα1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling

Significance In this study, we identify nerve-released glial cell line-derived neurotrophic factor (GDNF) family receptor (GFR)α1 as a key factor that enhances perineural invasion (PNI) through GDNF-Ret proto-oncogene (RET) signaling. We demonstrate that GFRα1 is released from nerves in a soluble form and cooperates with secreted GDNF to activate cancer cell surface RET, activating downstream signaling, cancer cell migration, and PNI. These findings advance our understanding of the molecular mechanisms of PNI and define the specific cancer cell requirements necessary for PNI to occur. This work promotes the concept that a ligand and receptor both released by the microenvironment may cooperate together to facilitate cancer invasion. These findings highlight the key participatory role that the nerve microenvironment plays in enabling cancer perineural invasion. The ability of cancer cells to invade along nerves is associated with aggressive disease and diminished patient survival rates. Perineural invasion (PNI) may be mediated by nerve secretion of glial cell line-derived neurotrophic factor (GDNF) attracting cancer cell migration through activation of cell surface Ret proto-oncogene (RET) receptors. GDNF family receptor (GFR)α1 acts as coreceptor with RET, with both required for response to GDNF. We demonstrate that GFRα1 released by nerves enhances PNI, even in the absence of cancer cell GFRα1 expression. Cancer cell migration toward GDNF, RET phosphorylation, and MAPK pathway activity are increased with exposure to soluble GFRα1 in a dose-dependent fashion. Dorsal root ganglia (DRG) release soluble GFRα1, which potentiates RET activation and cancer cell migration. In vitro DRG coculture assays of PNI show diminished PNI with DRG from GFRα1+/− mice compared with GFRα1+/+ mice. An in vivo murine model of PNI demonstrates that cancer cells lacking GFRα1 maintain an ability to invade nerves and impair nerve function, whereas those lacking RET lose this ability. A tissue microarray of human pancreatic ductal adenocarcinomas demonstrates wide variance of cancer cell GFRα1 expression, suggesting an alternate source of GFRα1 in PNI. These findings collectively demonstrate that GFRα1 released by nerves enhances PNI through GDNF-RET signaling and that GFRα1 expression by cancer cells enhances but is not required for PNI. These results advance a mechanistic understanding of PNI and implicate the nerve itself as a key facilitator of this adverse cancer cell behavior.

Poorly Differentiated Thyroid Carcinoma Presenting with Gross Extrathyroidal Extension: 1986–2009 Memorial Sloan-Kettering Cancer Center Experience

PURPOSE To describe the outcome of patients with poorly differentiated thyroid cancer (PDTC) presenting with gross extrathyroidal extension (ETE). MATERIALS AND METHODS After obtaining Institutional Review Board approval, we performed a retrospective review of a consecutive series of thyroid cancer patients treated by primary surgical resection with or without adjuvant therapy at Memorial Sloan-Kettering Cancer Center from 1986 to 2009. Out of 91 PDTC patients, 27 (30%) had gross ETE (T4a), and they formed the basis of our study. Of 27 patients, 52% were women. The median age was 70 years (range 27-87 years). Ten patients (37%) presented with distant metastases; four to bone, three to lung, and three to both bone and lung. All patients had extended total thyroidectomy, except two who had subtotal thyroidectomy. Twenty patients (74%) had central compartment neck dissection and 11 also had lateral neck dissection. Four patients had pN0, six (30%) pN1a, and 10 (50%) pN1b neck disease. Twenty-one patients (77%) had adjuvant therapy: 15 (55%) radioactive iodine (RAI) only, three (11%) postoperative external beam radiation (EBRT) only, and three (11%) had both RAI and EBRT. Overall survival (OS), disease-specific survival (DSS), local recurrence-free survival (LRFS), and regional recurrence-free survival (RRFS) were calculated by the Kaplan Meier method. RESULTS The median follow-up time was 57 months (range 1-197 months). The 5 year OS and DSS were 47% and 49%, respectively. This poor outcome was due to distant metastatic disease; 10 patients had distant metastases at presentation and a further six developed distant metastases during follow-up. Locoregional control was good with 5-year LRFS and RRFS of 70% and 62%, respectively. Overall, eight patients (30%) had recurrences: two had distant alone, two regional, two regional and distant, one local and distant, and one had local, regional, and distant recurrence. CONCLUSION Aggressive surgery in patients with PDTC showing gross ETE resulted in satisfactory locoregional control. Due to the small proportion of patients who received EBRT (22%), it is not possible to analyze its benefit on locoregional control. Of significance is the observation that the majority of patients (60%) who presented with or subsequently developed distant metastases eventually died of distant disease. New systemic therapies to target distant metastatic disease are required for improvements in outcome.

Radiation Impairs Perineural Invasion by Modulating the Nerve Microenvironment

Purpose Perineural invasion (PNI) by cancer cells is an ominous clinical event that is associated with increased local recurrence and poor prognosis. Although radiation therapy (RT) may be delivered along the course of an invaded nerve, the mechanisms through which radiation may potentially control PNI remain undefined. Experimental Design An in vitro co-culture system of dorsal root ganglia (DRG) and pancreatic cancer cells was used as a model of PNI. An in vivo murine sciatic nerve model was used to study how RT to nerve or cancer affects nerve invasion by cancer. Results Cancer cell invasion of the DRG was partially dependent on DRG secretion of glial-derived neurotrophic factor (GDNF). A single 4 Gy dose of radiation to the DRG alone, cultured with non-radiated cancer cells, significantly inhibited PNI and was associated with decreased GDNF secretion but intact DRG viability. Radiation of cancer cells alone, co-cultured with non-radiated nerves, inhibited PNI through predominantly compromised cancer cell viability. In a murine model of PNI, a single 8 Gy dose of radiation to the sciatic nerve prior to implantation of non-radiated cancer cells resulted in decreased GDNF expression, decreased PNI by imaging and histology, and preservation of sciatic nerve motor function. Conclusions Radiation may impair PNI through not only direct effects on cancer cell viability, but also an independent interruption of paracrine mechanisms underlying PNI. RT modulation of the nerve microenvironment may decrease PNI, and hold significant therapeutic implications for RT dosing and field design for patients with cancers exhibiting PNI.

Schwann cells induce cancer cell dispersion and invasion

Nerves enable cancer progression, as cancers have been shown to extend along nerves through the process of perineural invasion, which carries a poor prognosis. Furthermore, the innervation of some cancers promotes growth and metastases. It remains unclear, however, how nerves mechanistically contribute to cancer progression. Here, we demonstrated that Schwann cells promote cancer invasion through direct cancer cell contact. Histological evaluation of murine and human cancer specimens with perineural invasion uncovered a subpopulation of Schwann cells that associates with cancer cells. Coculture of cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner. Upon contact, Schwann cells induced the formation of cancer cell protrusions in their direction and intercalated between the cancer cells, leading to cancer cell dispersion. The formation of these processes was dependent on Schwann cell expression of neural cell adhesion molecule 1 (NCAM1) and ultimately promoted perineural invasion. Moreover, NCAM1-deficient mice showed decreased neural invasion and less paralysis. Such Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead exploited by cancer cells to promote perineural invasion and cancer progression.

The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.

Perineural invasion is a form of cancer progression where cancer cells invade along nerves. This behavior is associated with poor clinical outcomes; therefore, it is critical to identify novel ligand–receptor interactions between nerves and cancer cells that support the process of perineural invasion. A proteomic profiler chemokine array was used to screen for nerve-derived factors secreted from tissue explants of dorsal root ganglion (DRG), and CCL2 was identified as a lead candidate. Prostate cancer cell line expression of CCR2, the receptor to CCL2, correlated closely with MAPK and Akt pathway activity and cell migration towards CCL2 and DRG. In vitro nerve and cancer coculture invasion assays of perineural invasion demonstrated that cancer cell CCR2 expression facilitates perineural invasion. Perineural invasion is significantly diminished in coculture assays when using DRG harvested from CCL2−/− knockout mice as compared with control CCL2+/+ mice, indicating that CCR2 is required for perineural invasion in this murine model of perineural invasion. Furthermore, 20 of 21 (95%) patient specimens of prostate adenocarcinoma with perineural invasion exhibited CCR2 expression by immunohistochemistry, while just 3 of 13 (23%) lacking perineural invasion expressed CCR2. In summary, nerve-released CCL2 supports prostate cancer migration and perineural invasion though CCR2-mediated signaling. Implications: These results reveal CCL2–CCR2 signaling as a key ligand–receptor mechanism that mediates cancer cell communication with nerves during perineural invasion and highlight a potential future therapeutic target. Mol Cancer Res; 13(2); 380–90. ©2014 AACR.

Oncolytic vaccinia virus therapy of salivary gland carcinoma.

OBJECTIVE To examine the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) against a panel of 5 human salivary gland carcinoma cell lines. DESIGN The susceptibility of 5 salivary gland carcinoma cell lines to infection and oncolysis by GLV-1h68 was assessed in vitro and in vivo. RESULTS All 5 cell lines were susceptible to viral infection, transgene expression, and cytotoxic reactions. Three cell lines were exquisitely sensitive to infection by very low doses of GLV-1h68. Orthotopic parotid tumors exhibited more aggressive behavior compared with flank tumors. A single intratumoral injection of GLV-1h68 induced significant tumor regression without observed toxic effects in flank and parotid tumor models; controls demonstrated rapid tumor progression. CONCLUSION These promising results demonstrate significant oncolytic activity by an attenuated vaccinia virus for infecting and lysing salivary gland carcinomas, supporting future clinical trials.

Role of tracheal resection in thyroid cancer.

Purpose of review To provide a critical review of recent literature on the role of tracheal resection in thyroid cancer. Recent findings The current body of literature is centered on the controversy regarding how radical the extent of tracheal resection needs to be to achieve long-term control of thyroid carcinoma with tracheal invasion. Proponents of shave excision are guided by the reported survival outcomes comparable to segmental resections in a selected group of patients. Others believe that all patients should have a segmental sleeve resection to ensure clearance of transmural disease. Recent advances in microsurgical reconstruction may allow selected patients to undergo tracheal resection when a large tracheal defect is anticipated. Summary Tracheal invasion by well differentiated carcinoma is a marker of a more aggressive tumor behavior, defining a subpopulation of patients at a greater risk of recurrence and death. The goal of surgical intervention in this scenario is complete resection with no gross residual disease. A well designed prospective multi-institutional trial, taking into account depth of invasion, risk group stratification, histology, presence of distant metastasis, radioactive iodine trapping ability, adjuvant treatment, and long-term survival data, is needed to compare the outcomes following more conservative shave excision and segmental tracheal resection.

Murine model of neuromuscular electrical stimulation on squamous cell carcinoma: Potential implications for dysphagia therapy

Dysphagia is a potential consequence of treatment for head and neck cancer. Neuromuscular electrical stimulation (NMES) has evolved as a treatment option, with the goal of improved swallow function in patients with chronic dysphagia. However, the effects of NMES on tumorigenicity are unknown and often confound the initiation of this therapy, potentially limiting its efficacy in treating patients with head and neck cancer.

Surgical Decision Making in the Management of Well-Differentiated Thyroid Cancer

The incidence of thyroid cancer in the United States has been increasing. The biologic behavior of well-differentiated thyroid cancer (WDTC) can vary from indolent tumor to an aggressive disease with invasion of critical structures and/or widespread distant metastasis. Therefore, the risk stratification is crucial in understanding the biology of thyroid cancer, its prognosis and appropriate therapeutic interventions. In fact, understanding the nuances of biological behavior of thyroid carcinomas lays the foundation for the idea of selective surgical management of this disease.

Abstract 460: The role of Cdc42 in cancer cell perineural invasion

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Perineural invasion (PNI) is the ability of cancer cells to invade in, around, and along nerves. PNI is widely recognized as an adverse pathologic feature of many malignancies. Glial-derived neurotrophic factor (GDNF) is secreted by nerve and glial cells, and is important in neural development. We previously showed that GDNF activation of the RET/GFR-alpha tyrosine kinase in cancer cells induces chemotaxis towards nerves and PNI. Cdc42 is a member of the Rho family of G proteins and a key regulator of cell polarity. We demonstrate here that GDNF-mediated activation of RET/GFR-alpha leads to activation of Cdc42 as a mediator of cancer cell chemotaxis and PNI. Exposure of MiaPaCa2 cells to GDNF or dorsal root ganglia (DRG) activates Cdc42. SiRNA inhibition of Cdc42 impairs MiaPaCa2 chemotaxis towards GDNF or DRG in Boyden chamber assays. SiRNA inhibition of RET decreases Cdc42 activity under GDNF stimulation and MiaPaCa2 chemotaxis. To study dynamic interactions between nerves and cancer cells in vitro, we used a DRG and cancer cell co-culture model of PNI in matrigel. Analysis of individual cell trajectories reveals that cell velocity remains unchanged, while directional migration is disrupted with shRNA inhibition of Cdc42. The disruption of directional response impairs the ability of shRNA Cdc42 MiaPaCa2 cells to migrate along neurites, as visualized by time lapse microscopy and measured by area of PNI. MRI imaging of live mice with sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells reveals significantly diminished length and volume of PNI as compared with injected shRNA control cancer cells when matched for equivalent overall tumor volume. Murine sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells exhibited preserved hindlimb and hind paw neurologic function, and diminished perineural invasion by histopathologic examination as compared with shRNA control MiaPaCa2. To identify potential activators of Cdc42 in this model, we performed a comprehensive siRNA screen of guanine nucleotide exchange factors (GEFs), identifying specific GEFs which are necessary for MiaPaCa2 migration towards DRG in Boyden chambers. We identified and validated six GEFs ([FLJ10521][1], DOCK9, ARHGEF7, RASGRF1, ARHGEF5, PLEKHG1) that impair chemotaxis without affecting proliferation. These findings provide novel insights into the molecular mechanisms underlying cancer cell perineural invasion. The RET-GEF-Cdc42 axis may be an attractive target for novel therapies that may potentially interrupt perineural invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 460. doi:1538-7445.AM2012-460 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ10521&atom=%2Fcanres%2F72%2F8_Supplement%2F460.atom