Genetic disruption of ATAT1 causes RhoA downregulation through abnormal truncation of C/EBPß.

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPß is a major regulator of RHOA expression. Interestingly, the majority of C/EBPß in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPßp27) lacking the N-terminus of C/EBPß. Overexpression of a gene encoding a C/EBPßp27-mimicking protein via an N-terminal deletion in C/EBPß led to competitive binding with wild-type C/EBPß at the C/EBPß binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPßp27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPßp27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPßp27 in the nucleus through CTSL. We propose that CTSL and C/EBPßp27 may represent a novel therapeutic target for breast cancer treatment. [BMB Reports 2024; 57(6): 293-298].

Outcomes of bilateral axillo-breast approach robotic parathyroidectomy versus open parathyroidectomy for primary hyperparathyroidism: a single-institution retrospective study.

Purpose: Bilateral axillo-breast approach robotic parathyroidectomy (BABA-RP) aims to remove overactive or enlarged parathyroid glands with no visible neck collar incision. In this study, we compared the safety and surgical outcomes of BABA-RP vs. those of an open surgery group to ascertain whether BABA-RP is a safe and feasible surgical approach for patients with primary hyperparathyroidism (pHPT). Methods: This single-institution retrospective cohort study included 74 patients with primary HPT who underwent open parathyroidectomy (n = 37) or BABA-RP (n = 37) at our institution between November 2014 and March 2023. Patient demographics, biochemical cure rates, operative time, blood loss rates, and complication rates were examined and compared. Results: The patients in the BABA-RP group were younger and had a longer mean operative time. Regarding complication events, 2 patients in the open surgery group and 1 patient in the BABA-RP group had transient hypoparathyroidism. All 74 patients achieved biochemical cure at <6 months, regardless of the approach used. Two patients in the BABA-RP group and 1 patient in the open surgery group had carcinoma on surgical pathology. All 3 patients with parathyroid carcinoma remained recurrence-free at 1-year follow-up. Conclusion: Compared with the open procedure, BABA-RP is a safe and feasible procedure that provides an excellent biochemical cure rate for patients with pHPT and has superior cosmetic benefits with equivalent surgical outcomes.

EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.

Microtubule acetylation induced by oxidative stress regulates subcellular distribution of lysosomal vesicles for amyloid-beta secretion.

Excessive production and accumulation of amyloid-beta (Aß) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aß production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aß production and secretion by altering the subcellular distribution of Aß precursor protein (APP)-containing lysosomal vesicles. Under oxidative stress, both H4-APPSwe/Ind and HEK293T-APPSwe/Ind cell lines showed increased microtubule acetylation and Aß secretion. Knockdown (KD) of alpha-tubulin N-acetyltransferase 1 (ATAT1) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as ß-CTF and AICD, but also suppressed Aß secretion. Oxidative stress promoted the dispersion of LAMP1-positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein-mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aß secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aß plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress-induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aß secretion.

Downregulation of AP1S1 causes the lysosomal degradation of EGFR in non-small cell lung cancer.

Matrix stiffness has been shown to play a critical role in cancer progression by influencing various cellular processes, including epidermal growth factor (EGF) signaling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the role of adaptor-related protein complex 1 subunit sigma 1 (AP1S1), a component of adaptor protein complex-1, in the regulation of EGF receptor (EGFR) intracellular trafficking during cancer cell progression. We found that AP1S1 expression was upregulated under stiff matrix conditions, resulting in the regulation of EGFR trafficking in non-small cell lung adenocarcinoma cells. Knockout of AP1S1 caused the lysosomal degradation of EGFR, leading to suppressed EGF-induced anaplastic lymphoma receptor tyrosine kinase phosphorylation. In addition, the downregulation of AP1S1 increased the sensitivity of H1975 cancer cells, which are resistant to tyrosine kinase inhibitors, to erlotinib. Collectively, our results suggest that AP1S1 could regulate EGFR recycling under stiff matrix conditions, and AP1S1 inhibition could be a novel strategy for treating cancer cells resistant to EGFR-targeted anticancer drugs.

Bilateral axillo-breast approach robotic thyroidectomy in pediatric patients with thyroid disease and cancer.

PURPOSE: Robotic thyroid surgeries have cosmetic advantages over open surgeries, which are especially important in pediatric patients due to social stigmas from neck scars. The present study describes outcomes in a series of children who underwent bilateral axillo-breast approach (BABA) robotic thyroidectomy. METHODS: Pediatric patients aged ≤18 years who underwent BABA robotic thyroidectomy between 2014 and 2022 were retrospectively reviewed. Their clinical characteristics and surgical outcomes were evaluated. Surgical completeness was determined by comparing the number of retrieved lymph nodes per compartment to previously reported lymph node quantification. RESULTS: This study included 26 pediatric patients of mean age 15.5 years (range, 5-18 years). Of these 26 patients, 21 (80.8%) had thyroid cancer, with 9 (42.9%) having aggressive tumor pathology. The mean operation time was 157.3 min. No patient needed open conversion. Five patients (19.2%) experienced complications including transient hypoparathyroidism (n = 5), permanent hypoparathyroidism (n = 2), and chyle leakage (n = 1), but none experienced vocal cord palsy. The mean numbers of retrieved lymph nodes at levels IIa, III, IV, Vb, and VI were 3.2, 4.1, 5.6, 1.0, and 9.7, respectively, numbers comparable with the known quantification except for level II, as IIb dissection was omitted in our series. CONCLUSION: BABA robotic thyroidectomy is safe and effective in pediatric patients with thyroid diseases. Most complications were minor and transient, with the operation time similar to robotic thyroidectomy in adults. Surgical completeness was also satisfactory. Robotic thyroidectomy can be considered a surgical option, regardless of patient age or diagnosis.

Effect of body habitus on surgical outcomes following bilateral axillo-breast approach robotic thyroidectomy: a retrospective cohort study.

INTRODUCTION: BMI has been shown to predict perioperative outcomes in patients undergoing surgery. Most studies assessing the role of body habitus in thyroid surgery have focused on open surgery, with few studies assessing patients undergoing robotic surgery. The present study evaluated the effects of BMI on surgical outcomes in patients undergoing bilateral axillo-breast approach (BABA) robotic thyroidectomy. MATERIALS AND METHODS: This study included patients who underwent BABA robotic thyroidectomy between January 2013 and September 2021 at Seoul National University Bundang Hospital. Patients were categorized into six groups based on the WHO classification of overweight and obesity. Clinicopathological characteristics, postoperative complications, and surgical outcomes were evaluated. RESULTS: A total of 1921 patients were included. Comparisons of the six BMI groups showed no statistically significant differences in postoperative stay, resection margin involvement, postoperative complications, and recurrence. Subgroup analysis showed that hypocalcemia rates differed among BMI groups in patients who underwent lobectomy, with underweight and class II obese patients being at the highest risk ( P =0.006). However, the actual number of complications was relatively small and similar among the groups. In patients who underwent total thyroidectomy and isthmectomy, BMI was not correlated with postoperative complications, including hypocalcemia, recurrent laryngeal nerve palsy, postoperative bleeding, and chyle leakage. CONCLUSION: Body habitus was not significantly associated with operative time and postoperative complications in patients undergoing BABA robotic thyroidectomy, indicating that this approach is safe and feasible in obese patients.

BRAFV600E and TERT promoter C228T mutations on ThyroSeq v3 analysis of delayed skin metastasis from papillary thyroid cancer: a case report and literature review.

BACKGROUND: Skin metastasis from papillary thyroid cancer (PTC) is a rare entity that can occur up to decades after treatment of the primary tumor. Here, we present a patient who developed skin metastasis 10 years after treatment of her primary tumor and describe the molecular findings of the metastatic lesion. CASE PRESENTATION: A 44-year-old female with a history of PTC who underwent a total thyroidectomy and radioactive iodine (RAI) treatment 10 years ago presented with a 1.3-cm skin lesion along the prior thyroidectomy scar. A biopsy revealed metastatic PTC, and the patient underwent surgical excision of the lesion. ThyroSeq molecular testing showed the copresence of BRAFV600E mutation and TERT promoter C228T mutation. The patient subsequently received one round of adjuvant RAI therapy. CONCLUSIONS: A high index of suspicion is warranted in patients with a history of PTC who develop a skin lesion, even several years after remission of the primary disease. In patients with high-risk mutations, such as BRAFV600E and TERT promoter C228T mutations, long-term surveillance of disease recurrence is particularly important.

KPNA3 promotes epithelial-mesenchymal transition by regulating TGF-ß and AKT signaling pathways in MDA-MB-231, a triple-negative breast cancer cell line.

Karyopherin-α3 (KPNA3), a karyopherin- α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMTrelated genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-ß or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-ß-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type. [BMB Reports 2023; 56(2): 120-125].

Bilateral axillo-breast approach robotic total thyroidectomy in a 5-year-old patient with intractable Graves' disease.

Robotic thyroid surgeries in children are gaining popularity due to cosmetic advantages, but most previous studies include patients of school-age or older. We hereby introduce our case of a 5-year-old patient with Graves' disease to show key differences and similarities between pediatric and adult bilateral axillo-breast approach (BABA) robotic thyroidectomies. Despite the small body, no additional working space was needed and the flap creation was performed as similarly done in adults. The anatomy was not much different, except that the parathyroid tissues were easily identified due to low body fat, and the prominent thymus covered most of the level VI area. The patient did not experience postoperative complications such as hypoparathyroidism or vocal cord palsy. Postoperative wounds showed excellent results with minimal scars. BABA robotic thyroidectomy can be performed safely in pediatric patients and may be considered an alternative option for conventional open thyroidectomy in children.

How Effective is the Use of Molecular Testing in Preoperative Decision Making for Management of Indeterminate Thyroid Nodules?

INTRODUCTION: We performed Thyroseq v2 molecular testing on indeterminate thyroid nodules and evaluated whether they underwent a management change from the standard of thyroid lobectomy. METHODS: We conducted a retrospective analysis of all indeterminate thyroid nodules that underwent Thyroseq v2 molecular testing from 2014 to 2019 at a large academic center. Pathology was reviewed by thyroid cytopathologists. Thyroseq results were reported benign (malignancy probability less than 10%) or suspicious (malignancy probability greater than 30%). The primary endpoint was a management change from a diagnostic lobectomy. RESULTS: A total of 142 nodules were included: 113 (80%) Bethesda III and 29 (20%) Bethesda IV. Seventy-three nodules underwent surgical management and 69 did not. We noted a change in management in 64% (91/142) of nodules. Patients who underwent a change in management to no surgery had a significantly higher rate of benign Thyroseq result than those without a change (75.8% vs. 49.0%, p = 0.001). On logistic regression analysis, a benign Thyroseq result was a positive independent predictor of a change to no surgery (OR 3.87, 95% CI 1.69-8.89). Nodule size, multiple nodules, compressive symptoms, and history of hypothyroidism were not significant. Of the 91 patients who underwent a management change, 71% (65/91) did not undergo surgery. On follow-up (average 985 ± 615 days), 12% (8/65) of those nodules were growing or developed suspicious features requiring surgery. CONCLUSIONS: Molecular testing helped avoid surgery in almost half our population with indeterminate thyroid nodules, and benign results may help avoid surgery in asymptomatic patients with indeterminate thyroid nodules.

Prevalence of Postoperatively Detected High-risk Features in 2- to 4-cm Papillary Thyroid Cancers.

CONTEXT: The 2015 American Thyroid Association guidelines proposed thyroid lobectomy as an acceptable option for 1- to 4-cm papillary thyroid cancers (PTC) without extrathyroidal extension (ETE) or lymph node (LN) metastasis. However, high-risk features are often detected postoperatively, even in tumors that are considered low risk on preoperative workup. A continued evaluation is necessary to determine the optimal treatment strategies. OBJECTIVE: We examined the frequency of preoperatively and postoperatively detected high-risk features in 2- to 4-cm PTCs to assess the appropriate surgical extent. METHODS: All patients who underwent a thyroid surgery between 2015 and 2020 with a final diagnosis of 2- to 4-cm PTC were selected. Demographics, preoperative findings, perioperative course, and surgical pathology were retrospectively analyzed. RESULTS: Of the entire study cohort (N = 424), 244 (57.5%) patients had at least 1 of the following high-risk features: gross ETE (18.6%), distant metastasis (1.2%), >3 LN involvement with extranodal extension (24.8%), any LN > 3 cm (0.5%), positive margin (13.2%), TERT mutation (2.6%), vascular invasion (10.8%), cN1 disease (28.5%), and > 5 LN involvement (30.4%). Two hundred patients had neither ETE nor LN metastasis on preoperative imaging, but 62/200 (31.0%) were found to have at least 1 of the aforementioned high-risk features on final pathology. Preoperative imaging had sensitivities of 75.9% and 44.4% for detecting gross ETE and LN metastasis, respectively. CONCLUSION: A significant portion of patients with 2- to 4-cm PTCs, including those who preoperatively met the criteria for lobectomy, were found to have high-risk features on final pathology. Careful patient selection and appropriate counseling are necessary when considering lobectomy for tumors greater than 2 cm.

The short video lecture for robotic bilateral axillo-breast approach to lateral neck lymph node dissection.

Lateral neck lymph node dissection (LND) along with total thyroidectomy is the standard treatment for thyroid cancer patients with metastases to the lateral neck lymph nodes. In general, lateral neck LND removes lymph nodes located at levels II to V ipsilateral to the thyroid cancer and preserves the spinal accessory nerve, internal jugular vein, and sternomastoid muscle during surgery. This video article was written to introduce the robotic bilateral axillo-breast approach for lateral neck LND and to describe the surgical method.

Adipocyte HIF2α functions as a thermostat via PKA Cα regulation in beige adipocytes.

Thermogenic adipocytes generate heat to maintain body temperature against hypothermia in response to cold. Although tight regulation of thermogenesis is required to prevent energy sources depletion, the molecular details that tune thermogenesis are not thoroughly understood. Here, we demonstrate that adipocyte hypoxia-inducible factor α (HIFα) plays a key role in calibrating thermogenic function upon cold and re-warming. In beige adipocytes, HIFα attenuates protein kinase A (PKA) activity, leading to suppression of thermogenic activity. Mechanistically, HIF2α suppresses PKA activity by inducing miR-3085-3p expression to downregulate PKA catalytic subunit α (PKA Cα). Ablation of adipocyte HIF2α stimulates retention of beige adipocytes, accompanied by increased PKA Cα during re-warming after cold stimuli. Moreover, administration of miR-3085-3p promotes beige-to-white transition via downregulation of PKA Cα and mitochondrial abundance in adipocyte HIF2α deficient mice. Collectively, these findings suggest that HIF2α-dependent PKA regulation plays an important role as a thermostat through dynamic remodeling of beige adipocytes.

Identifying influential subpopulations in metapopulation epidemic models using message-passing theory.

Identifying influential subpopulations in metapopulation epidemic models has far-reaching potential implications for surveillance and intervention policies of a global pandemic. However, there is a lack of methods to determine influential nodes in metapopulation models based on a rigorous mathematical background. In this study, we derive the message-passing theory for metapopulation modeling and propose a method to determine influential spreaders. Based on our analysis, we identify the most dangerous city as a potential seed of a pandemic when applied to real-world data. Moreover, we particularly assess the relative importance of various sources of heterogeneity at the subpopulation level, e.g., the number of connections and mobility patterns, to determine properties of spreading processes. We validate our theory with extensive numerical simulations on empirical and synthetic networks considering various mobility and transmission probabilities. We confirm that our theory can accurately predict influential subpopulations in metapopulation models.

The Role of Surgeon-Performed Office and Preincision Ultrasounds in Localization of Parathyroid Adenomas in Primary Hyperparathyroidism.

OBJECTIVE: We studied the use of surgeon-performed office ultrasound (OU) and preincision ultrasound (PIU) in preoperatively localizing parathyroid adenomas in primary hyperparathyroidism (PHPT). METHODS: A retrospective chart review was performed for patients with PHPT who underwent parathyroidectomy between 2013 and 2015. The results of OU and PIU were recorded and compared with the final surgical pathology. RESULTS: Of 348 patients with PHPT, 285 (81.9%) had single-lesion disease, 49 (14.1%) had double-lesion disease, and 14 (4.0%) had multigland disease with 3 or more lesions. For single-lesion disease, the overall sensitivity and specificity of OU to correctly lateralize the lesion were 64.2% and 91.2%, while those of PIU were 89.4% and 93.6%, respectively. The sensitivity and specificity of PIU were comparable to those of 4-dimensional computed tomography (87.1% and 90.7%, respectively) and 99mTc-sestamibi scintigraphy (70.4% and 95.9%, respectively). While the majority of PIU cases were preceded by other imaging studies, the accuracy in localizing lesions was not largely affected by the presence of prior computed tomography and/or 99mTc-sestamibi scintigraphy, as opposed to ultrasounds only. For detecting the presence of multigland disease, the sensitivity and specificity of OU were 26% and 92.2%, while those of PIU were 64.3% and 94.7%, respectively. CONCLUSION: Surgeon-performed OU and PIU are valuable tools in preoperatively localizing the parathyroid adenoma in single-lesion disease, while their utility may be limited for double-lesion or multigland disease. PIU in particular yields high accuracy in detecting parathyroid lesions in combination with other imaging modalities.

Casein kinase 2 promotes the TGF-ß-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix.

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-ß-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-ß-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, significantly abrogated the TGF-ß-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-ß-induced activation of α-TAT1 in a soft matrix. [BMB Reports 2022; 55(4): 192-197].

Double transitions and hysteresis in heterogeneous contagion processes.

In many real-world contagion phenomena, the number of contacts to spreading entities for adoption varies for different individuals. Therefore, we study a model of contagion dynamics with heterogeneous adoption thresholds. We derive mean-field equations for the fraction of adopted nodes and obtain phase diagrams in terms of the transmission probability and fraction of nodes requiring multiple contacts for adoption. We find a double phase transition exhibiting a continuous transition and a subsequent discontinuous jump in the fraction of adopted nodes because of the heterogeneity in adoption thresholds. Additionally, we observe hysteresis curves in the fraction of adopted nodes owing to adopted nodes in the densely connected core in a network.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics.

PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.