Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Pre-operative hyperfractionated concurrent radiochemotherapy for locally advanced rectal cancers: a phase II clinical study.

OBJECTIVE: The study was prospectively designed as a single-arm, single-institution prospective trial of pre-operative concomitant hyperfractionated radiotherapy (HART) with co-administration of chemotherapy based on 5-fluorouracil (5FU) in patients with T2/N+ or T3/any N resectable mid-low primary rectal cancer. The aim of the study was to assess the safety and efficacy of accelerated HART with concurrent 5FU-based chemotherapy in patients with locally advanced rectal cancer. METHODS: Patients with resectable locally advanced (≥T3 or N+) rectal cancer were eligible. The patients received total dose 42 Gy in 28 fractions of 1.5 Gy, two times daily, with at least 8 h of interval, with concurrent chemotherapy: 325 mg m-2 of 5FU (bolus) on Days 1-3 and Days 16-18 (except for cN0 patients for whom only one cycle on Days 1-3 was prescribed). The primary end point included tolerance, post-operative complication rate and pathological response rate. The secondary end points included locoregional relapse-free survival, metastasis-free survival and overall survival. RESULTS: Out of 53 enrolled patients; 2 did not undergo surgery. Of the 51 patients evaluable for pathological response, there were 8 (15.6%), 20 (39.3%), 18 (35.3%) and 5 (9.8%) patients with tumour regression grade 0, 1, 2 and 3, respectively. Downstaging of the primary tumour and lymph nodes was observed in 22 (43%) and 25 (49%) patients, respectively. The primary tumour ypCR (ypT0) rate was 15% (8/51). The nodal ypCR rate for cN+ patients was 60% (21/35). The total ypCR (ypT0N0M0) rate was 11% (6/51). Toxicity included: Grade 3 diarrhoea (4/51, 7.8%), Grade 2 diarrhoea (22/51, 43.1%), Grade 2 leukopenia (7/51, 13.7%), Grade 2 neutropenia (6/51, 11.7%) and Grade 1 thrombocytopenia (3/51, 5.9%). No Grade 4 toxicity was reported. Nine patients (18%) presented with post-operative complications (during the 3 months after surgery). There were 6 locoregional relapses (11.8%) and distant metastasis occurred in 11 patients (21.6%). The 2-year cumulative locoregional relapse-free survival, metastasis-free survival and overall survival was 87%, 79% and 89%, respectively. CONCLUSION: The proposed pre-operative HART with co-administration of 5FU had acceptable toxicity profile and provided satisfactory rate of ypCR. This created rationale to initiate a Phase III randomized study that was registered under ClinicalTrials.gov Identifier: NCT01814969. Advances in knowledge: The results of this research show that responders to pre-operative radiochemotherapy have favourable outcome. Tumour regression grade as prognostic clinical feature holds the promise of better classifying patients at high risk of local and systemic recurrence and this issue may be an interesting objective for future research.

Cell type-dependent modulation of the gene encoding heat shock protein HSPA2 by hypoxia-inducible factor HIF-1: Down-regulation in keratinocytes and up-regulation in HeLa cells.

HSPA2 belongs to the multigene HSPA family, whose members encode chaperone proteins. Although expression and function of HSPA2 is mainly associated with spermatogenesis, recent studies demonstrated that in humans, the gene is active in various cancers, as well as in normal tissues, albeit in a cell type-specific manner. In the epidermis, HSPA2 is expressed in keratinocytes in the basal layer. Currently, the mechanisms underlying the regulation of HSPA2 expression remain unknown. This study was aimed at determining whether HIF-1 and its binding site, the hypoxia-response element (HRE) located in the HSPA2 promoter, are involved in HSPA2 regulation. As a model system, we used an immortal human keratinocyte line (HaCaT) and cervical cancer cells (HeLa) grown under control or hypoxic conditions. Using an in vitro gene reporter assay, we demonstrated that in keratinocytes HSPA2 promoter activity is reduced under conditions that facilitate stabilization of HIF-1α, whereas HIF-1 inhibitors abrogated the suppressive effect of hypoxia on promoter activity. Chromatin immunoprecipitation revealed that HIF-1α binds to the HSPA2 promoter. In keratinocytes, hypoxia or overexpression of a stable form of HIF-1α attenuated the expression of endogenous HSPA2, whereas targeted repression of HIF-1α by RNAi increased transcription of HSPA2 under hypoxia. Conversely, in HeLa cells, HSPA2 expression increased under conditions that stimulated HIF-1α activity, whereas inhibition of HIF-1α abrogated hypoxia-induced up-regulation of HSPA2 expression. Taken together, our results demonstrate that HIF-1 can exert differential, cell context-dependent regulatory control of the HSPA2 gene. Additionally, we also showed that HSPA2 expression can be stimulated during hypoxia/reoxygenation stress.

Evaluation of outcomes and treatment safety of patients with metastatic colorectal cancer to the liver with estimation of prognostic factors.

UNLABELLED: Liver resection is essential part of colorectal cancer liver metastases (CLM) treatment. Mean 5-year overall survival after resection achieves 30-45%. There are many factors influencing long-term outcomes, and among them the inflammatory response to tumor plays an important role. THE AIM OF THE STUDY: was evaluation of outcomes and treatment safety of patients with metastatic colorectal cancer to the liver with estimation of prognostic factors. MATERIAL AND METHODS: 130 consecutive patients (70 men and 60 women) operated in MSC Institute and Cancer Center in Gliwice from 2001 to 2009 due to colorectal liver metastases were analysed. Age of the patients ranged from 33 to 82 years (median 60 years). 96 (74%) patients underwent potentially radical resection, and in remaining 34 (26%) was performed radiofrequency ablation (RFA) alone or combined with the resection. In the resection group 37 right hepatectomies, 11 left hepatectomies, 28 segmentectomies and 20 metastasectomies were performed. Disease-free survival (DFS) and overall survival (OS) were statistically analysed using the Kaplan-Meier method. Factors determining DFS and OS were analysed using Cox regression model. RESULTS: In the resection group the 3- and 5-years OS was 64,5% and 46,6% respectively, and the 3- and 5-years DFS was 32% and 30,5% respectively. In the RFA group the 3- and 5-years OS was 33% and 9,5%. Statistically significant prognostic factors in the resection group in uni- and multivariate analysis were: grade and nodal involvement of the primary tumor, diameter of metastatic focus, positive and narrow (<1 mm) resection margins, preoperative fibrinogen level, preoperative neutrophil to lymphocyte ratio and leukocyte amount of the peripheral blood. The perioperative mortality rate was 3%. CONCLUSIONS: Liver resection due to colorectal liver metastases is a safe and effective method resulting in high survival rates. We confirmed some generally accepted prognostic factors influencing longterm outcomes and shown the impact of inflammatory response. We also confirmed the hypothesis that preoperative plasma fibrinogen level influences outcomes after liver resection due to CLM.

Neoadjuvant treatment for unresectable rectal cancer: an interim analysis of a multicentre randomized study.

PURPOSE: To present an interim analysis of the trial comparing two neoadjuvant therapies for unresectable rectal cancer. METHODS: Patients with fixed cT3 or cT4 or locally recurrent rectal cancer without distant metastases were randomized to either 5 × 5 Gy and 3 courses of FOLFOX4 (schedule I) or 50.4 Gy delivered in 28 fractions given simultaneously with 5-Fu, leucovorin and oxaliplatin (schedule II). Surgery in both groups was performed 12 weeks after the beginning of radiation and 6 weeks after neoadjuvant treatment. RESULTS: 49 patients were treated according to schedule I and 48 according to schedule II. Grade III+ acute toxicity was observed in 26% of patients in group I and in 25% in group II. There were two toxic deaths, both in group II. The microscopically radical resection (primary endpoint) rate was 73% in group I and 71% in group II. Overall and severe postoperative complications were recorded in 27% and 9% of patients vs. 16% and 7%, respectively. Pathological complete response was observed in 21% of the patients in group I and in 9% in group II. CONCLUSIONS: The interim analysis revealed no major differences in acute toxicity and local efficacy between the two evaluated strategies.

Moderately low alpha/beta ratio for rectal cancer may best explain the outcome of three fractionation schedules of preoperative radiotherapy.

PURPOSE: To estimate the alpha/beta ratio for rectal cancer according to the outcome of three fractionation schedules of preoperative radiotherapy. METHODS AND MATERIALS: Between 1996 and 2002, 168 patients with locally advanced rectal cancer were treated as follows: 53 patients received 25 Gy in 5 Gy per fraction, 45 received 30 Gy in 3.0 Gy per fraction, and 70 were treated with accelerated hyperfractionation (42 Gy, 1.5 Gy per fraction, given twice daily). No patients received concurrent chemotherapy. The clinical characteristics of the groups were comparable. Surgery was performed shortly after radiotherapy. Crude data on locoregional tumor control were fitted directly using a linear-quadratic model, and the actuarial data were analyzed using Cox model. RESULTS: A linear-quadratic model provided an alpha estimate of 0.339 (SE 0.115) and beta estimate of 0.067 (SE 0.027), which resulted in an alpha/beta ratio of 5.06 Gy (95% confidence interval -0.1 to 10.3). In all three schemes the overall radiation treatment time was short, which limits the rationales for incorporating time effect into the model. If, however, time was incorporated the alpha/beta ratio was 11.1 Gy and the dose increment required to compensate for repopulation was 0.15 Gy/day. The actuarial analysis provided similar alpha/beta estimates. CONCLUSION: Although because of the retrospective character of the study, nonrandomized selection of fractionation schedule, and uncontrolled quality of surgery the present results can be regarded as hypothesis generating only, the control rates obtained in the pelvis are consistent with a moderately low alpha/beta ratio for rectal cancer.

[Influence of prognostic factors on the risk of recurrence in breast cancer patients after radical treatment]. / Wplyw czynników rokowniczych na niepowodzenia po radykalnym leczeniu chorych na raka piersi.

The analysis includes 730 consecutive breast cancer patients operated radically. The impact on local and distant recurrences as well as the probability of 5-year disease free survival according to age, hormonal status, primary tumor and axillary nodes stage, number of involved nodes, presence of extracapsular invasion and histopathological grade was assessed. Results of this analysis showed the stage of axillary lymph nodes as the most important prognostic factor for the estimation of the risk of failure. It was also revealed that well-known, classic factors are often insufficient to determine appropriate prognosis.