p27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer.

In colorectal cancer, mutation of KRAS (RASMUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.

Preclinical vascular alterations in obese adolescents detected by Laser-Doppler Flowmetry technique.

BACKGROUND AND AIMS: Childhood obesity promotes adverse changes in cardiovascular structure and function. This study evaluated whether alterations in skin microcirculation were already present in obese adolescents in a pre-clinical phase of cardiovascular disease. METHODS AND RESULTS: After an overnight fasting 22 obese adolescents and 24 normal-weight controls of similar age and gender distribution underwent clinical and blood examination and assessment of microvascular function by using two non-invasive techniques such as Peripheral Artery Tonometry (PAT) and Laser-Doppler Flowmetry (LDF). As compared to normal weight subjects, obese children had higher blood pressure, were significantly more hyper-insulinemic and insulin resistant, showing significantly higher plasma total cholesterol, LDL cholesterol, triglycerides and alanine aminotransferase (ALT). LDF showed lower pre- and post-occlusion forearm skin perfusion (perfusion units/second (PU/sec); median [IQR]) in obese than in normal weight subjects (pre-occlusion: 1633.8 [1023.5] vs. 2281.1 [1344.2]; p = 0.015. Post-occlusion: 4811.3 [4068.9] vs. 7072.8 [7298.8]; p = 0.021), while PAT revealed similar values of reactive hyperemia index (RHI). In entire population, fat mass % (FM%) was an independent determinant of both pre-and post-occlusion skin perfusion. Finally, being obese was associated with a higher risk to have a reduction of both pre- and post-occlusion skin perfusion (OR = 5,82 and 9,27, respectively). CONCLUSION: LDF showed very early, pre-clinical, vascular involvement in obese adolescents, characterized by impaired skin microcirculation, possibly reflecting a more diffuse microvascular dysfunction to other body tissues. Whether changing life style and improving weight may reverse such pre-clinical alterations remains to be established.

miRNAs as Candidate Biomarker for the Accurate Detection of Atypical Endometrial Hyperplasia/Endometrial Intraepithelial Neoplasia.

Endometrial cancer is the most common gynecologic malignancy in developed countries. Estrogen-dependent tumors (type I, endometrioid) account for 80% of cases and non-estrogen-dependent (type II, non-endometrioid) account for the rest. Endometrial cancer type I is generally thought to develop via precursor lesions along with the increasing accumulation of molecular genetic alterations. Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia is the least common type of hyperplasia but it is the type most likely to progress to type I cancer, whereas endometrial hyperplasia without atypia rarely progresses to carcinoma. MicroRNAs are a class of small, non-coding, single-stranded RNAs that negatively regulate gene expression mainly binding to 3'-untranslated region of target mRNAs. In the current study, we identified a microRNAs signature (miR-205, miR-146a, miR-1260b) able to discriminate between atypical and typical endometrial hyperplasia in two independent cohorts of patients. The identification of molecular markers that can distinguish between these two distinct pathological conditions is considered to be highly useful for the clinical management of patients because hyperplasia with an atypical change is associated with a higher risk of developing cancer. We show that the combination of miR-205, -146a, and -1260b has the best predictive power in discriminating these two conditions (>90%). With the aim to find a biological role for these three microRNAs, we focused our attention on a common putative target involved in endometrial carcinogenesis: the oncosuppressor gene SMAD4. We showed that miRs-146a,-205, and-1260b directly target SMAD4 and their enforced expression induced proliferation and migration of Endometrioid Cancer derived cell lines, Hec1a cells. These data suggest that microRNAs-mediated impairment of the TGF-ß pathway, due to inhibition of its effector molecule SMAD4, is a relevant molecular alteration in endometrial carcinoma development. Our findings show a potential diagnostic role of this microRNAs signature for the accurate diagnosis of Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia and improve the understanding of their pivotal role in SMAD4 regulation.

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.

Dynamic markers based on blood perfusion fluctuations for selecting skin melanocytic lesions for biopsy.

Skin malignant melanoma is a highly angiogenic cancer, necessitating early diagnosis for positive prognosis. The current diagnostic standard of biopsy and histological examination inevitably leads to many unnecessary invasive excisions. Here, we propose a non-invasive method of identification of melanoma based on blood flow dynamics. We consider a wide frequency range from 0.005-2 Hz associated with both local vascular regulation and effects of cardiac pulsation. Combining uniquely the power of oscillations associated with individual physiological processes we obtain a marker which distinguishes between melanoma and atypical nevi with sensitivity of 100% and specificity of 90.9%. The method reveals valuable functional information about the melanoma microenvironment. It also provides the means for simple, accurate, in vivo distinction between malignant melanoma and atypical nevi, and may lead to a substantial reduction in the number of biopsies currently undertaken.

Peripheral microvascular dysfunction as an independent predictor of atherosclerotic damage in type 1 diabetes patients: a preliminary study.

It has been recently hypothesized that peripheral microvascular dysfunction may contribute to atherosclerotic damage (AD) in diabetic patients. In order to test this hypothesis, we assessed forearm skin post-occlusive reactive hyperaemia (skin-PORH), an index of peripheral microvascular function, using laser-Doppler flowmetry, in 40 type 1 diabetes patients (T1D-pts), aged 49 ± 11 years, with no known cardiovascular complications, and in 50 age and sex-matched healthy control subjects (CS). T1D-pts also underwent carotid arteries ultrasound scanning (Ca-US) and ankle-brachial index (ABI) measurement. An arbitrary index of AD (AD-index), ranging from "0" (normal ABI, normal Ca-US) to "3" (abnormal ABI, one or more plaques at the Ca-US), was determined in T1D-pts. Linear and multiple regression analyses were performed to identify independent predictors of AD in T1D-pts. T1D-pts had a lower skin-PORH compared with CS (p = 0.015). In T1D-pts AD-index resulted to be negatively related with skin-PORH (R = 0.44; p < 0.005) or deep-breathing test (DBT) (R = 0.53; p < 0.0005), and positively related with systolic arterial pressure (R = 0.31; p < 0.05), microalbuminuria (R = 0.46; p < 0.005), patients' age (R = 0.51; p < 0.001) and diabetes duration (R = 0.39; p < 0.05). At the multiple regression analysis skin-PORH (R = 0.36; p < 0.005), patients' age R = 0.24; p < 0.05) and DBT (R = 0.4 - p < 0.005) resulted to be independent predictors of AD-index in T1D-pts. These preliminary findings support the hypothesis that peripheral microvascular dysfunction may contribute to AD in T1D-pts.

Assessment of skin microvascular endothelial function in patients with acute unilateral vestibular syndrome.

Abnormalities in labyrinth vasculature, resulting in labyrinth ischemia may be responsible for acute unilateral vestibular syndrome (AVS). However, since no tools for the study of the labyrinth microvasculature are available in clinical settings, labyrinth microvascular abnormalities in AVS patients (AVS-pts) can only be hypothesized on the basis of the their cardiovascular risk profile. Considering that skin microcirculation may mirror vascular function in other body districts, we examined skin endothelial function in 20AVS-pts and in 20 healthy control subjects (CS), with the aim of predicting labyrinth microvascular abnormalities in the same AVS-pts, potentially involved in the pathogenesis of their AVS. AVS-pts and CS underwent laser-Doppler flowmetry measurement of the skin forearm vasodilator response (SVR) to iontophoresis of the endothelial-dependent vasodilator acetylcholine (ACh) and to the endothelial-independent vasodilator sodium nitroprusside (SNP). SVR to ACh was significantly lower than to SNP in AVS patients (p < 0.005, ANOVA for repeated measures), consistent with skin endothelial dysfunction, while no significant differences in SVR between ACh and SNP were observed in CS. Accordingly with an arbitrary cut-off of 30% or greater reduction in SVR to ACh compared to SNP, endothelial dysfunction was found in 4 (20%) of CS, and in 14 (70%) of AVS-pts (6 with associated co-morbidities potentially responsible for endothelial dysfunction, and 8 without these co-morbodities). This study shows that the investigation of skin endothelial function in AVS-pts may be helpful in identifying AVS-pts in whom an ischemic origin of AVS might be more probable, in spite of their low cardiovascular risk profile.

Study of skin vasomotion in type 1 diabetic patients and of its possible relationship with clinical and laboratory variables.

Vascular oscillation (vasomotion) occurs in the microcirculation and is thought to be a significant contributor to tissue perfusion. Our aim was to assess skin vasomotion (SV) of type 1 diabetic patients (T1D-pts) and its relationship with clinical or laboratory variables of the studied T1D-pts. Forearm endothelial-, sympathetic- and myogenic-dependent SV were assessed basally and after 3 min of forearm ischemia in 40 T1D-pts and 50 healthy controls, by spectral analysis of laser-Doppler (LD) signal at the frequency ranges of 0.009-0.02 Hz, 0.021-0.06 Hz and 0.061-0.2 Hz, respectively. Post-ischemic per cent increase (PI%-increase) in power spectral density (PSD) of skin endothelial- and sympathetic-dependent VS was significantly reduced in T1D-pts compared to controls (p < 0.0005, p < 0.0001, respectively). Linear regression analysis showed a significant positive relationship between PI%-increase of endothelial-dependent SV and heart rate variation during laying-standing test (R = 0.65, p = 0.00001), and a negative relationship between PI%-increase in PSD of skin LD signal 0.009-1.6 Hz spectrum and glycated haemoglobin serum levels (R = 0.44, p = 0.0036) in T1D-pts. These results are consistent with reduced skin endothelial- and sympathetic-dependent stimulated SV and with relationships between some clinical or laboratory variables and SV parameters in the T1D-pts studied.

Subcutaneous adipose tissue blood flow and vasomotion in morbidly obese patients: long term effect of gastric bypass surgery.

Since recent findings suggest a relationship between reduction in adipose tissue blood flow (ATBF) and metabolic or vascular complications in obese patients (Ob-pts), increase in ATBF may be considered as a further goal in the treatment of obesity, besides fat mass reduction. Therefore, this preliminary study aimed at assess subcutaneous ATBF and vasomotion in morbidly obese patients and whether sustained weight loss induced by Roux-en-Y gastric bypass (RYGB) affects the same parameters. Using laser-Doppler flowmetry (LDF) and spectral Fourier analysis, subcutaneous ATBF was measured and subcutaneous ATBF oscillations (ATBF-O) were analyzed - within three frequency intervals related to vasomotion - in 16 Ob-pts, before and about one year after RYGB, and in 10 lean, healthy control subjects (CS). Before RYGB, Ob-Pts showed an important reduction in subcutaneous ATBF compared to CS (4.8 ± 2.7 PU vs 79.9 ± 34.5 PU, respectively; p < 0.0001), as well as higher normalized power spectral density (N-PSD) values of subcutaneous ATBF-O, - related to vasomotion. One year after RYGB, sustained weight loss in Ob-pts was associated with a slight but significant increase in subcutaneous ATBF (10.0 ± 6.6 PU, p < 0.05) and with almost complete normalization in N-PSD values of ATBF-O, related to vasomotion, compared to before RYGB. The slight subcutaneous ATBF increase, we observed in Ob-pts after sustained weight loss, moves toward a desirable goal. This finding suggests verifying whether an even more sustained weight loss in Ob-pts could determine a greater increase in subcutaneous ATBF and/or, more importantly, it could also determine a significant increase in visceral ATBF.

Investigation of skin vasoreactivity and blood flow oscillations in hypertensive patients: effect of short-term antihypertensive treatment.

BACKGROUND AND METHOD: In order to evaluate whether arterial hypertension (AH) affects skin microcirculation, 46 newly diagnosed, never-treated, hypertensive patients and 20 healthy normotensive controls underwent a forearm skin postocclusive reactive hyperaemia (PORH) test, using laser-Doppler flowmetry (LDF). Their resting skin blood flow oscillations (SBFOs) were also investigated using wavelet spectral analyses of skin LDF tracings within six frequency subintervals in the 0.005-2 Hz spectral range. To evaluate whether antihypertensive treatment affects skin microcirculation, the same measurements were repeated in 22 of the recruited hypertensive patients after 8 ±â€Š2 weeks of antihypertensive treatment. RESULTS: Significantly reduced PORH, together with significantly higher spectral amplitudes within the majority of the investigated SBFO subintervals, was found in untreated hypertensive patients compared with controls. In the 22 hypertensive patients who completed the follow-up, there was a significant increase in PORH after antihypertensive treatment compared with before (357 ±â€Š178 vs. 284 ±â€Š214%, respectively, P < 0.05). Following antihypertensive treatment, the same 22 hypertensive patients did not differ significantly from controls either in PORH or in the majority of the investigated SBFO frequency subintervals. CONCLUSION: This study showed reduced skin vasoreactivity in the hypertensive patients, confirming that antihypertensive treatment negatively affects skin microcirculation. The short period of efficacious antihypertensive treatment resulted in normalization of skin vasoreactivity in hypertensive patients who completed the follow-up, suggesting that antihypertensive treatment affects positively skin microcirculation in AH. The SBFO increase in untreated hypertensive patients, and its almost complete normalization in treated hypertensive patients, suggests that SBFO enhancement in untreated hypertensive patients could be an adaptive reversible response to AH.

Skin vasodilator function and vasomotion in patients with morbid obesity: effects of gastric bypass surgery.

Obesity-associated microvascular dysfunction (MVD) involves different body tissues, including skin, and concurs to increased cardiovascular risk in obese patients (Ob-P). Generalized improvement of MVD is an important goal in obesity treatment. Since skin MVD mirrors generalized systemic MVD, skin microvascular investigation in prospective studies in Ob-P may surrogate microvascular investigation in organs more important for cardiovascular risk of the studied patients. In this prospective study, we measured forearm skin post-occlusive reactive hyperaemia (PORH), as percentage flow increase from baseline, and skin vasomotion in 37 Ob-P before Roux-en-Y gastric bypass (RYGB), and in 24 of them about 1 year after RYGB, using laser Doppler flowmetry (LDF). The spectral contribution of skin LDF signal oscillations in the frequency intervals of 0.01-0.02 Hz, 0.02-0.06 Hz, and 0.06-0.2 Hz--corresponding to endothelial-, sympathetic-, and myogenic-dependent vasomotion, respectively, was measured by means of spectral Fourier analysis. The same measurements were also performed in 28 healthy, lean subjects (HLS). Before RYGB, Ob-P had a significant reduction in PORH and in the all vasomotion parameters investigated, compared with HLS. After RYGB, Ob-P who completed the follow-up, had a significant weight loss (∼40 kg on average), together with a full normalisation in PORH and in vasomotion parameters, regardless of diabetes status. Surgically induced sustained weight loss resulted in full normalisation of skin microvascolar function in Ob-P about 1 year after RYGB. This result suggests a beneficial effect of sustained weight loss on generalized MVD of the studied Ob-P.