Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade.

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

The role of atorvastatin in regulating the immune response leading to vascular damage in a model of Kawasaki disease.

Superantigens have been implicated in a number of diseases including Kawasaki disease (KD), a multi-system vasculitis resulting in coronary artery aneurysms. We have characterized a murine disease model in which coronary arteritis is induced by a novel superantigen found in Lactobacillus casei cell wall extract (LCWE). Using this animal model of KD, we have identified three pathogenic steps leading to coronary artery aneurysm formation. These steps include T cell activation and proliferation, production of the proinflammatory cytokine tumour necrosis factor (TNF)-α and up-regulation of matrix metalloproteinase 9 (MMP-9), an elastolytic protease. In addition to their cholesterol-lowering effects, 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors (statins) have pleotropic immunomodulatory properties. Thus, we examined the effect of atorvastatin in modulating each of these three critical pathogenic processes leading to aneurysm formation in the disease model. Atorvastatin inhibited lymphocyte proliferation in response to superantigen stimulation in a dose-dependent manner. This inhibition was also observed for production of soluble mediators of inflammation including interleukin (IL)-2 and TNF-α. The inhibitory effect on proliferation was rescued completely by mevalonic acid, confirming that the mechanism responsible for this inhibitory activity on immune activation was inhibition of HMG-CoA reductase. Similarly, TNF-α-induced MMP-9 production was reduced in a dose-dependent manner in response to atorvastatin. Inhibition of extracellular-regulated kinase (ERK) phosphorylation appears to be the mechanism responsible for inhibition of MMP-9 production. In conclusion, atorvastatin is able to inhibit critical steps known to be important in the development of coronary aneurysms, suggesting that statins may have therapeutic benefit in patients with KD.

Inhibition of matrix metalloproteinase-9 activity improves coronary outcome in an animal model of Kawasaki disease.

Kawasaki disease (KD) is the leading cause of acquired heart disease of children in North America. It is characterized by a massive immune activation and multi-system vasculitis, which evolves into a site-specific inflammatory response focused at the coronary arteries. Coronary artery (CA) inflammation leads to elastin breakdown, destruction of the vessel wall and aneurysm formation. We have demonstrated recently the pivotal role of tumour necrosis factor (TNF)-alpha-mediated matrix metalloproteinase (MMP)-9 activity in the pathogenesis of elastin breakdown in a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis. Using this model, we evaluated the in vitro effects of doxycycline, an antibiotic with MMP inhibitory function, in modulating key pathogenic stages of disease leading to CA damage. Doxycycline inhibits T cell activation and TNF-alpha production in peripheral immune cells, as assessed by thymidine incorporation and a TNF bioassay respectively. Additionally, doxycycline inhibits directly MMP-9 enzymatic activity derived from TNF-alpha-stimulated vascular smooth muscle cells as assayed by zymography. More importantly, in vivo treatment of Lactobacillus casei cell wall extract (LCWE)-injected mice with doxycycline reduces significantly the incidence of CA elastin breakdown and reduces loss of elastin. Therefore, doxycycline can mitigate TNF-alpha-induced MMP-9-mediated coronary elastin breakdown and improve coronary outcome. Agents with the ability to inhibit both inflammation and the downstream effects of inflammation, such as MMP-9 activity, offer a promising therapeutic strategy for the management of children with KD.

Lessons learned from an animal model of Kawasaki disease.

Kawasaki disease is the most common cause of multisystem vasculitis in childhood. Kawasaki disease has been reported throughout the world and affects children of all ethnicity. Coronary artery damage from Kawasaki disease is the leading cause of acquired heart disease in children in the developed world. Diagnostic tests and prognostic markers are lacking, and questions remain unanswered in our understanding of the etiopathogenesis of the disease, thus limiting our ability to improve therapy and coronary outcome. In this article I will review advances made in an animal model of disease, which has helped advance our understanding of the etiology and pathogenesis of this fascinating clinical syndrome.

Hospital preparedness for chemical and biological incidents in Hong Kong.

The risk of mass exposure to toxic substances has increased steadily during the twentieth century due to the expansion of industry and the deliberate development and use of agents of chemical warfare. Although Hong Kong is considered a relatively safe place, hoax anthrax attacks have occurred since 17 October 2001. People who have been seriously injured by hazardous materials have a greater chance of recovery without complications when appropriate emergency treatments are provided. Recognition and identification of hazardous materials, assessment of the conditions, decontamination, and protection of staff and facilities are important elements in the formulation of a contingency plan. The objective of this article is to outline the efforts of the Hong Kong Hospital Authority in formulating a hospital response to incidents involving hazardous materials.

Genetic identification of a locus, Mot1, that affects renal tumor size in the rat.

Prognosis and treatment of solid tumors are directly dependent on the stage of disease. For any type of cancer, tumor characteristics such as size, multiplicity, and metastatic potential are highly heterogeneous among patients. Our understanding of the genetic determinants of tumor burden is rudimentary. Here, rats carrying a germline mutation of the gene Tsc2 were found to develop variable size and number of renal tumors. We hypothesize that "modifier" genes unlinked to Tsc2 affect its expressivity. Using a backcross (BC) analysis between the two strains that showed the greatest difference in tumor size (Fischer344 and Brown Norway), we mapped a quantitative trait locus based on tumor volume to rat chromosome 3q, lying in the interval between D3Mit3 and D3Rat17, with a maximum lod score of 4.4. This locus, Mot1 (modifier of Tsc2 1), accounts for approximately 35% of the genetic variation in tumor size between the two strains. No significant difference in tumor multiplicity was noted between Brown Norway and Fischer344 rats. This suggests that Mot1 modulates the rate of disease progression and not tumor initiation. Candidate genes on rat chromosome 3 included Tsc1, whose product interacts biochemically with the TSC2 protein, but it was excluded on the basis of linkage analysis (LOD=0.01). Comparative genomics suggest that the Mot1 region is represented by human chromosomes 15q and 20pq. Our results provide the first evidence of a modifier gene affecting the Tsc2 pathway in the progression of renal tumorigenesis.

Loss of tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disorder associated with an alteration of the TSC2 tumor suppressor gene which encodes for the protein product tuberin. The disease is characterized by the development of hamartomas, e.g. cutaneous angiofibromas which consist of vascular cells, interstitial cells, and normal components of the skin. The Eker rat model, an animal model of inherited cancer, has been shown to carry a mutation of TSC2. METHODS: Immunohistochemical analyses of human angiofibromas were performed using antibodies directed against tuberin and angiogenic growth factors. Proliferation of human dermal microvascular endothelial cells (HDMEC) was determined after incubation with the supernatants of TSC2 (+/+) and TSC2 (-/-) rat embryonic fibroblasts (REF) that were derived from the Eker strain. RESULTS: Loss of the expression of tuberin was observed in the interstitial cells of 13 of 39 angiofibromas. The expression of tuberin was retained in the vascular cells. In all analyzed angiofibromas, the angiogenic factors bFGF, PD-ECGF, VEGF and angiogenin were detected in the interstitial cells and/or vascular cells. Expression of PDGF-B and TGF-beta1 was weak. Tissue culture supernatants from TSC2 (-/-) REF stimulated the growth of HDMEC significantly more than supernatants from TSC2 (+/+) REF. CONCLUSION: A functional loss of tuberin may stimulate vascular growth.

Axillary web syndrome after axillary dissection.

BACKGROUND: Some patients undergoing axillary lymph node dissection (ALND) experience postoperative pain and limited range of motion associated with a palpable web of tissue extending from the axilla into the ipsilateral arm. The purpose of this study is to characterize the previously undescribed axillary web syndrome (AWS). METHODS: To identify patients with AWS, a retrospective review was performed of all invasive breast cancer patients treated by a single surgeon (REM) between 1980 and 1996. Records were also reviewed of 4 more recent patients who developed AWS after undergoing sentinel node lymph node dissection (SLND) without ALND. RESULTS: Among 750 sequentially treated patients, 44 (6%) developed AWS between 1 and 8 weeks after their axillary procedure. The palpable subcutaneous cords extended from the axillary crease down the ipsilateral arm, across the antecubital space, and in severe cases down to the base of the thumb. The web was associated with pain and limited shoulder abduction (< or = 90 degrees in 74% of patients). AWS resolved in all cases within 2 to 3 months. AWS also occurred after SLND. Tissue sampling of webs in 4 patients showed occlusion in lymphatic and venous channels. CONCLUSIONS: AWS is a self-limiting cause of morbidity in the early postoperative period. More limited axillary surgery, with less lymphovenous disruption, might reduce the severity and incidence of this syndrome, although SLND does not eliminate its occurrence.

Internal mammary lymph node drainage patterns in patients with breast cancer documented by breast lymphoscintigraphy.

BACKGROUND: Metastases to internal mammary lymph nodes (IMN) may occur in patients with breast cancer and may alter treatment recommendations. The purpose of this study was to identify the frequency of IMN drainage in patients undergoing breast lymphoscintigraphy and sentinel lymph node dissection (SLND). METHODS: The combined technique of peritumoral injection of radiocolloid and Lymphazurin blue for SLND was performed on 220 patients. All patients underwent preoperative lymphoscintigraphy before SLND. Lesion location by quadrant included: 110 upper outer (UOQ), 49 lower outer (LOQ), 30 upper inner (UIQ), 24 lower inner (LIQ), and 7 central. RESULTS: Drainage to any nodal basin was observed in 184 of 220 patients (84%). IMN drainage was documented in 37 of 220 (17%) of patients. IMN drainage without evidence of axillary drainage occurred in 2 of 220 patients(1%). Drainage to the IMN based on quadrant location of the lesion was as follows: UOQ, 10%; LOQ, 27%; UIQ, 17%; LIQ, 25%; and central, 29%. CONCLUSIONS: Internal mammary lymph node drainage shown by breast lymphoscintigraphy is common. Tumors in all quadrants may drain to IMNs, although drainage is significantly more common from quadrants other than the UOQ. Further studies are needed to determine whether lymphoscintigraphy drainage patterns identify patients at the highest risk for IMN metastases who may benefit from radiotherapy.

Tuberin phosphorylation regulates its interaction with hamartin. Two proteins involved in tuberous sclerosis.

Hamartin and tuberin are products of the tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a characteristic spectrum of tumor-like growths develop resulting in the syndrome of tuberous sclerosis complex. The phenotypes associated with TSC1 and TSC2 mutations are largely indistinguishable suggesting a common biochemical pathway. Indeed, hamartin and tuberin have been shown to interact stably in vitro and in vivo. Factors that regulate their interaction are likely critical to the understanding of disease pathogenesis. In this study, we showed that tuberin is phosphorylated at serine and tyrosine residues in response to serum and other factors, and it undergoes serial phosphorylation that can be detected by differences in electrophoretic mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth inhibitory effects of tuberin when overexpressed in COS1 cells, consistent with a loss of function mutation. Examination of a second pathologic mutation, P1675L, revealed a similar relationship between limited phosphorylation and reduced interaction with hamartin. Our data show for the first time that 1) tuberin is phosphorylated at tyrosine and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin phosphorylation, and 3) defective phosphorylation of tuberin is associated with loss of its tumor suppressor activity. These findings suggest that phosphorylation may be a key regulatory mechanism controlling TSC1-TSC2 function.

Children with morphea have normal self-perception.

We evaluated the self-esteem and quality of life of 47 children with morphea with the use of the Harter self-perception profile for children and Visual Analog Scale. Most children with morphea have normal self-worth and a high quality of life. Morphea, like some other childhood chronic illnesses, does not impair self-esteem.

Methotrexate and corticosteroid therapy for pediatric localized scleroderma.

INTRODUCTION: Localized scleroderma (LS) can cause permanent disability, and there is no universally accepted effective treatment. Methotrexate (MTX) has been shown to be effective in the treatment of systemic sclerosis. OBJECTIVES: To determine the efficacy and tolerability of MTX and corticosteroid therapy in patients with LS. METHODS: MTX, 0.3 to 0.6 mg/kg per week, was given to 10 patients (6 girls, 4 boys; mean age, 6.8 years; mean disease duration before starting treatment, 4 years) with active LS. In addition, pulse intravenous methylprednisolone, 30 mg/kg for 3 days monthly for 3 months, was given to 9 patients at the initiation of therapy. RESULTS: One patient discontinued taking MTX after a month; the remaining 9 patients responded. The median time to response was 3 months (95% CI, 1.15-4.85). One responder discontinued taking MTX after a year because of leukopenia; the LS worsened within 2 months. In another patient LS flared up after 10 months and responded to an increased dose of MTX and intravenous methylprednisolone. At the last follow-up visit, all patients who continued to receive MTX therapy had inactive skin lesions. CONCLUSION: Treatment with MTX and corticosteroids appears to be effective in the treatment of LS and is generally well tolerated. A placebo-controlled study is necessary to confirm the efficacy of MTX therapy in LS.

Increased false negative sentinel node biopsy rates after preoperative chemotherapy for invasive breast carcinoma.

BACKGROUND: Sentinel lymph node dissection (SLND) has been a promising new technique in breast carcinoma staging, but could be unreliable in certain patient subsets. The current study assessed whether age, preoperative chemotherapy, tumor size, and/or previous excisional biopsy influenced the identification of sentinel nodes (SLNs) or the reliability of a node-negative SLND in predicting a node negative axilla. METHODS: Eighty-two patients who had clinically negative axillae underwent SLND followed by Level I/II axillary lymph node dissection (ALND). SLNDs were performed using both technetium-99m (Tc-99m) labeled colloid and isosulfan blue dye. SLNs were analyzed by hematoxlyin and eosin and immunocytochemical techniques. RESULTS: SLNs were successfully identified in 80% of patients. Mapping success was decreased among postmenopausal women but was not influenced by preoperative chemotherapy, large tumor size, or previous excisional biopsy. Of the 31 successfully mapped, node positive patients, 5 had false negative (FN) SLNDs (overall FN rate = 16%). Of the 9 successfully mapped patients who had received preoperative chemotherapy and had positive axillary nodes, 3 had FN SLND (FN rate = 33%). The presence of clinically positive lymph nodes before chemotherapy did not predict which patients would have a subsequent FN SLND. T3 tumor size, but not previous excision, was associated significantly with increased FN rate, although the FN rate for previous excision was 11%. No FN SLND occurred with T1/T2 tumors that were not excised previously and had not received preoperative chemotherapy. CONCLUSIONS: Preoperative chemotherapy was associated with an unacceptably high FN rate for SLND. While larger tumor size also was associated with FN SLND, this effect might have been due to preoperative chemotherapy use in these patients. Small sample size precluded determining whether excisional biopsy before mapping increased FN SLND rates independently.

Sentinel lymph node mapping for breast cancer: analysis in a diverse patient group.

PURPOSE: To evaluate sentinel lymph node mapping in patients with breast cancer. MATERIALS AND METHODS: Sixty-two patients with breast cancer scheduled to undergo axillary nodal dissection underwent scintigraphic localization of sentinel lymph nodes with filtered technetium 99m sulfur colloid. At surgery, isosulfan blue was injected. Sentinel nodes were identifiable by blue color and by radioactivity with hand-held gamma probe. Results were analyzed statistically. RESULTS: A sentinel lymph node was identified in 49 patients (79%). Lymph nodes were positive for metastatic disease in 26 patients (42%). The mapping success rate was 78% (n = 21) in the 27 patients with no prior surgery, 78% (n = 18) in the 23 patients with prior surgery, and 86% (n = 12) in the 14 patients with prior chemotherapy. Axillary nodes were positive in 11 (41%) of the 27 patients with no prior intervention, six (26%) of the 23 patients with prior surgery, and 10 (71%) of the 14 patients with prior chemotherapy. There were no false-negative findings in patients without prior intervention. Four patients with positive nodes had false-negative sentinel nodes. CONCLUSION: Sentinel lymph node mapping and biopsy without axillary dissection is appropriate in patients with breast cancer who have not undergone prior intervention. Further study is necessary to ascertain the accuracy of the procedure for patients who have undergone presurgical chemotherapy or previous excisional biopsy.

Technical aspects of sentinel node lymphoscintigraphy for breast cancer.

OBJECTIVE: A significant morbidity risk is associated with axillary nodal dissections for breast cancer. Many treatment decisions are based on axillary nodal status. Lymphatic mapping and sentinel node biopsy have been investigated to determine if the histology of the sentinel node reflects the remaining lymph node basin. We describe the technical aspects of sentinel node lymphoscintigraphy for breast cancer. METHODS: Ninety-three patients had lymphoscintigraphy for breast cancer. Patients with palpable lesions had 4 concentric injections around the site and lesions requiring localization had injections made through tubing connected to the localizing wire introducer needle. Immediate static images were acquired and the sentinel node was marked for surgery. Marks were reverified using a handheld gamma probe. RESULTS: Lymph nodes were visualized by lymphoscintigraphy in 87% of cases. Time to visualization of lymph nodes ranged from 1-120 min with a mean of 28 min. An average of 1.5 nodes were visualized. The overall success rate for identifying the sentinel node at time of surgery was 85%. CONCLUSION: We conclude that lymphoscintigraphy for breast cancer is a detailed procedure that requires coordination with radiology and surgery teams to ensure proper identification of sentinel lymph nodes.

Generation of humanized mice susceptible to peptide-induced inflammatory heart disease.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death. In certain mouse major histocompatibility complex (MHC) backgrounds, myocarditis and inflammatory cardiomyopathy can be triggered by immunization with heart muscle-specific proteins. Similarly, chronic heart disease in humans has been linked to certain HLA alleles, such as HLA-DQ6. However, there is no experimental evidence showing that human MHC class II molecules and peptides derived from human proteins are involved in the pathogenesis of myocarditis and DCM. METHODS AND RESULTS: We generated double CD4- and CD8-deficient mice transgenic for human CD4 (hCD4) and human HLA-DQ6 to specifically reconstitute the human CD4/DQ6 arm of the immune system in mice. Transgenic hCD4 and HLA-DQ6 expression rendered genetically resistant C57BL/6 mice susceptible to the induction of autoimmune myocarditis induced by immunization with cardiac myosin. Moreover, we identified heart-specific peptides derived from both mouse and human alpha-myosin heavy chains capable of inducing inflammatory heart disease in hCD4 and HLA-DQ6 double transgenic mice but not in hCD4 single transgenic littermates. The autoimmune inflammatory heart disease induced by the human heart muscle-specific peptide in hCD4 and HLA-DQ6 double transgenic mice shared functional and phenotypic features with the disease occurring in disease-susceptible nontransgenic mice. CONCLUSIONS: Our data provide the first genetic and functional evidence that human MHC class II molecules and a human alpha-myosin heavy chain-derived peptide can cause inflammatory heart disease and suggest that human inflammatory cardiomyopathy can be caused by organ-specific autoimmunity. The humanized mice generated in this study will be an ideal animal model to further elucidate the pathogenesis of inflammatory heart disease and facilitate the development of rational treatment strategies.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development.

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function.